TW202337474A - Irna compositions and methods for silencing angiotensinogen (agt) - Google Patents

Abstract

The present invention relates to RNAi agents, e.g., double stranded RNA (dsRNA) agents, targeting an angiotensinogen (AGT) gene. The invention also relates to methods of using such RNAi agents to inhibit expression of an AGT gene and to methods of preventing and treating an AGT-associated disorder, e.g., hypertension.

Description

iRNA compositions and methods for silencing angiotensinogen (AGT) Related applications

This application claims priority from U.S. Provisional Application No. 63/229085, filed on August 4, 2021, and U.S. Provisional Application No. 63/272769, filed on October 28, 2021. The entire contents of each of the above applications are incorporated herein by reference.

The present invention relates to RNAi agents, and in particular to methods of using such RNAi agents to inhibit AGT gene expression and methods of preventing and treating AGT-related disorders.

The renin-angiotocin-aldosterone system (RAAS) plays an important role in blood pressure regulation. The RAAS cascade begins with the release of provasotocin from the liver and the release of renin from the juxtaglomerular cells of the kidney into the circulation. Renin secretion is stimulated by a variety of factors, including Na+ load in the distal tubule, β-sympathetic nerve stimulation, or reduced renal perfusion. Active renin in the plasma cleaves angiotensinogen (produced by the liver) to angiotensin I, which is then converted to angiotensin II by circulating and locally expressed angiotensin-converting enzyme (ACE). Most of the effects of angiotensin II on the RAAS are exerted through its binding to the angiotensin II type 1 receptor (AT ₁ R), resulting in arterial vasoconstriction, tubular and glomerular effects such as enhancement of Na+ Reabsorption or regulation of glomerular filtration rate. Furthermore, in conjunction with other stimuli such as adrenocorticotropin, antidiuretic hormones, catecholamines, endothelin, serotonin, and Mg2+ and K+ levels, AT ₁ R stimulation leads to aldosterone release, which in turn alternately promotes Na+ and K+ in the renal distal tubule of excretion.

Dysregulation of the RAAS results in, for example, excessive angiotensin II or AT ₁ R stimulation leading to hypertension, which can lead to, for example, increased oxidative stress in the heart, kidneys, and arteries, promote inflammation, hypertrophy, and fibrosis, and lead to , for example, left ventricular fibrosis, arterial remodeling, and glomerulosclerosis.

Hypertension is the most common and controllable disease in developed countries, affecting 20 to 50% of the adult population. Hypertension is a major risk factor for a variety of diseases, disorders, and conditions, such as shortened life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysms (e.g., aortic aneurysm), peripheral arterial disease, cardiac injury (e.g., cardiac enlargement or hypertrophy) and other cardiovascular-related diseases, disorders or conditions. In addition, hypertension has been shown to be an important risk factor for cardiovascular disease morbidity and mortality, accounting for or contributing to 62% of all strokes and 49% of all cases of heart disease. In 2017, changes to the guidelines for the diagnosis, prevention, and treatment of hypertension were developed, which provide goals for reducing the risk of further developing hypertension-related diseases and disorders associated with hypertension (see e.g., Reboussin et al. Systematic Review for the 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the Prevention,Detection,Evaluation,and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology /American Heart Association Task Force on Clinical Practice Guidelines. J Am Coll Cardiol. 2017 Nov 7.pii: S0735-1097(17)41517-8.doi: 10.1016/j.jacc.2017.11.004; and Whelton et al. ( 2017ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. J Am Coll Cardiol. 2017 Nov 7.pii:S0735-1097(17)41519-1.doi:10.1016/j.jacc.2017.11.006).

Despite the large number of antihypertensive drugs available for the treatment of hypertension, more than two-thirds of individuals are not controlled by one antihypertensive drug and require two or more antihypertensive drugs selected from different drug classes. This further reduces the number of individuals with controlled blood pressure, as compliance decreases and side effects increase with the number of medications.

The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript encoding the angiotensinogen (AGT) gene. The AGT gene may be intracellular, for example, within a cell of an individual, such as a human individual. The present invention also provides methods of using the iRNA compositions of the present invention for inhibiting the expression of the AGT gene and/or for treating individuals who would benefit from inhibiting or reducing the expression of the AGT gene, for example, those who suffer from or are prone to suffer from and AGT-related disorders, such as hypertension.

Accordingly, in one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting angiotensinogen (AGT) expression in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region shares, of which the rights shares include at least 15, for example, 15, 16, 17, 18, 19 or 20 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 by no more than 0, 1, 2 or 3 nucleotides and the antisense strand contains at least 15 , for example, 15, 16, 17, 18, 19 or 20 consecutive nucleotides that differ from the corresponding part of SEQ ID NO: 2 or SEQ ID NO: 4 by no more than 1, 2 or 3 nucleotides.

In another aspect, the invention provides double-stranded ribonucleic acid (dsRNA) for inhibiting angiotensinogen (AGT) expression in a cell, wherein the dsRNA includes a sense strand and an antisense strand forming a double-stranded region, wherein the dsRNA The antisense strand includes a complementary region to the mRNA encoding AGT, wherein the complementary region includes at least 15, such as 15, 16, 17, 18, 19 or 20 antisense to any one of any one from Tables 2 to 7 Consecutive nucleotides whose nucleotide sequences differ by no more than 0, 1, 2, or 3 nucleotides.

In one embodiment, the dsRNA agent contains a sense strand that contains at least 15 contiguous nucleotides and differs by no more than 3 nucleotides from any nucleotide sequence of the sense strand in any one of Tables 2 to 7. strands, and antisense strands containing at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any nucleotide sequence of the antisense strand in any of Tables 2 to 7.

In one embodiment, the dsRNA agent contains a sense strand that contains at least 15 contiguous nucleotides and differs by no more than 2 nucleotides from any nucleotide sequence of the sense strand in any one of Tables 2 to 7. strands, and antisense strands containing at least 15 consecutive nucleotides that differ by no more than 2 nucleotides from any nucleotide sequence of the antisense strand in any of Tables 2 to 7.

In one embodiment, the dsRNA agent contains a sense strand that contains at least 15 contiguous nucleotides and differs by no more than 1 nucleotide from any nucleotide sequence of the sense strand in any one of Tables 2 to 7. strands, and antisense strands containing at least 15 contiguous nucleotides that differ by no more than 1 nucleotide from any nucleotide sequence of the antisense strand in any of Tables 2 to 7.

In one embodiment, the dsRNA agent contains a sense strand comprising or consisting of a nucleotide sequence selected from the group consisting of the nucleotide sequence of the sense strand in any one of Tables 2 to 7; and An antisense strand comprising or consisting of a nucleotide sequence selected from the group consisting of the nucleotide sequence of the antisense strand in any one of Tables 2 to 7.

In one embodiment, the dsRNA agent includes at least one modified nucleotide.

In one embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides; substantially all of the nucleotides in the antisense strand are modified nucleotides; or substantially all of the nucleotides in the sense strand are modified nucleotides. All nucleotides in the sense strand and substantially all nucleotides in the antisense strand are modified nucleotides.

In one embodiment, all nucleotides of the sense strand are modified nucleotides; all nucleotides of the antisense strand are modified nucleotides; or all nucleotides of the sense strand are modified nucleotides. The nucleotides and all nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the at least one modified nucleotide is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'- O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides , restricted ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkanes base-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, N-methyl Phinoline nucleotides, aminophosphates, unnatural bases including nucleotides, tetrahydropyran-modified nucleotides, 1,5-hydrohexitol-modified nucleotides, cyclohexenyl-modified Nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, vinylphosphonate nucleotides, 5'-phosphate containing nucleotides, containing Nucleotides that are 5'-phosphate mimetics, thermally destabilized nucleotides, glycol-modified nucleotides (GNA), 2'-phosphate-containing nucleotides, and 2-O-(N-methylethyl amide) modified nucleotides; and combinations thereof.

In one embodiment, the at least one modified nucleotide is selected from the group consisting of: LNA, HNA, CeNA, 2' -methoxyethyl, 2' - O-alkyl, 2 ' -O-allyl, 2' - C-allyl, 2' -fluoro, 2' -deoxy, 2'-hydroxy and ethylene glycol; and combinations thereof.

In one embodiment, the at least one modified nucleotide is selected from the group consisting of: deoxy-nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro Modified nucleotides, 2'-deoxy-modified nucleotides, glycol-modified nucleotides (GNA), e.g., Ggn, Cgn, Tgn or Agn, nucleotides with 2' phosphate, e.g. G2p, C2p, A2p or U2p, and nucleotides containing phosphorothioate groups, and combinations thereof.

In another embodiment, the at least one modified nucleotide is a nucleotide having a thermally labile nucleotide modification.

In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of: no base modification; mismatch with the opposite nucleotide in the duplex; destabilizing sugar modification, 2 '-Deoxy-modified, acyclic nucleotides, unlocked nucleic acids (UNA) and glyceryl nucleic acids (GNA).

In some embodiments, the modified nucleotides comprise a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the dsRNA agent contains 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3'-terminus of one strand. Where necessary, the shares are anti-anti shares. In another implementation form, the shares are equity shares. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5'-terminus of a strand. Optionally, the stock is an antisense stock. In another implementation form, the shares are equity shares. In another embodiment, the phosphorothioate or the methylphosphonate core The inter-glycoside linkages are located at both the 5' and 3'-termini of one strand. Where necessary, the shares are anti-anti shares. In another implementation form, the shares are equity shares.

The double-stranded region may be 19 to 30 nucleotide pairs in length; 19 to 25 nucleotide pairs in length; 19 to 23 nucleotide pairs in length; 23 to 27 nucleotide pairs in length; or 21 to 23 nucleotide pairs in length.

In one embodiment, each strand independently is no more than 30 nucleotides in length.

In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

The complementary region may be at least 17 nucleotides in length; between 19 and 23 nucleotides in length; or 19 nucleotides in length.

In one embodiment, the at least one strand includes a 3' overhang of at least 1 nucleotide. In another embodiment, the at least one strand includes a 3' overhang of at least 2 nucleotides.

In one embodiment, the dsRNA agent includes a ligand.

In one embodiment, the ligand is coupled to the 3' end of the sense strand of the dsRNA agent

In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

In one embodiment, the ligand is one or more GalNAc derivatives attached via monovalent, divalent or trivalent branched linkers.

In one embodiment, the ligand is

In one embodiment, the dsRNA agent is conjugated to the ligand as follows:

And, where X is O or S.

In one embodiment, the X is O.

In one embodiment, the dsRNA agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3'-terminus of a strand, e.g., an antisense or sense strand.

In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5'-terminus of a strand, e.g., an antisense or sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at both the 5'- and 3'-termini of a strand. In one implementation, the stock is an anti-contra stock.

In one embodiment, the base pair at position 1 at the 5' -end of the antisense strand of the duplex is an AU base pair.

The present invention also provides cells containing any dsRNA agent of the present invention and pharmaceutical compositions containing any dsRNA agent of the present invention.

The pharmaceutical composition of the present invention may comprise the dsRNA agent in an unbuffered solution, for example, saline or water, or the pharmaceutical composition of the present invention may comprise the dsRNA agent in a buffered solution, for example, comprising acetate, citrate, Buffered solutions of prolamins, carbonates or phosphates or any combination thereof; or phosphate buffered saline (PBS).

In one aspect, the invention provides methods of inhibiting angiotensinogen (AGT) gene expression in a cell. The method includes contacting the cells with any dsRNA agent of the invention or any pharmaceutical composition of the disclosure, thereby inhibiting the expression of the AGT gene in the cells.

In one embodiment, the cell line is used in an individual, e.g., a human subject, e.g., an individual with angiotensinogen (AGT)-related disorders, such as high blodd pressure, hypertension, borderline Hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, Renovascular hypertension, Goldblatt hypertension, intraocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, Arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, Stroke, kidney disease, renal failure, systemic sclerosis disease, intrauterine growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, Type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome.

In some embodiments, the subject has a systolic blood pressure of at least 130 mm Hg or a diastolic blood pressure of at least 80 mm Hg. In certain embodiments, the subject has a systolic blood pressure of at least 140 mm Hg and a diastolic blood pressure of at least 80 mm Hg. In some embodiments, the system is part of a population susceptible to salt sensitivity, is overweight, is obese, or is pregnant.

In some embodiments, the cells are contacted with a dsRNA agent to inhibit at least 50%, 60%, 70%, 80%, 90%, 95% of AGT expression (e.g., AGT expression prior to initial contact with the dsRNA agent). concentration; for example, in subjects administered the first dose of the dsRNA agent). In some embodiments, AGT expression is inhibited to reduce AGT protein levels in the individual's serum by at least 50%, 60%, 70%, 80%, 90%, or 95%, e.g., prior to initial contact with the cell with a dsRNA agent. AGT performance concentration compared.

In one aspect, the present invention provides methods of treating an individual suffering from a disorder who would benefit from reduced expression of angiotensinogen (AGT). The method involves administering to an individual a therapeutically effective amount of any dsRNA of the invention or any pharmaceutical composition of the present disclosure, thereby treating the individual having the disorder and who would benefit from reduced expression of angiotensinogen (AGT).

In another aspect, the present invention provides a method of preventing at least one symptom in an individual suffering from a disorder who would benefit from reduced expression of angiotensinogen (AGT). The method comprises administering to an individual a prophylactically effective amount of any dsRNA of the present invention or any pharmaceutical composition of the present invention, such that in an individual who has a disorder and would benefit from a reduction in provasotocin (AGT) expression Prevent at least one symptom.

In some embodiments, the disorder is a provasotocin-related disorder.

In some embodiments, the subject has a systolic blood pressure of at least 130 mm Hg or a diastolic blood pressure of at least 80 mm Hg. In certain embodiments, the subject has a systolic blood pressure of at least 140 mm Hg and a diastolic blood pressure of at least 80 mm Hg. In some implementations, the system is human. In some embodiments, the system is part of a population susceptible to salt sensitivity, is overweight, is obese, or is pregnant.

In some implementations, the AGT-related disorder is selected from the group consisting of: high blood pressure, hypertension, borderline hypertension, essential hypertension, secondary hypertension , isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, Glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic Heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, kidney disease, renal failure, systemic sclerosis, Intrauterine fetal growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes mellitus), metabolic syndrome.

In a further aspect, the present invention also provides methods of inhibiting the expression of angiotensinogen (AGT) in an individual. The method involves administering to an individual a therapeutically effective dose of any dsRNA as described herein, thereby inhibiting AGT expression in the individual.

In one implementation, the system is human.

In one embodiment, administration of the dsRNA agent to an individual results in a reduction in AGT protein accumulation in the individual.

In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to 50 mg/kg.

In one embodiment, the dsRNA agent is administered to the individual subcutaneously.

In one embodiment, the method of the present invention further includes determining the AGT magnitude in the individual sample as the AGT protein concentration in the blood or serum or urine or liver tissue sample.

In some embodiments, the methods of the present invention further comprise determining the individual's bradylin level; protonin level, or blood pressure.

In some embodiments, the methods of the invention further comprise administering to the individual an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is selected from the group consisting of: diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vasodilators Agents, calcium channel blockers, aldosterone antagonists, α2-agonists, renin inhibitors, α-blockers, peripherally acting adrenergic drugs, selective D1 receptor partial agonists, non-selective α -Adrenergic antagonists, synthetic and steroid mineralocorticoids; or combinations of any of the foregoing, as well as therapeutic agents for hypertension in formulated combinations. In some embodiments, the additional therapeutic agent includes an angiotensin II receptor antagonist, such as losartan, valsartan, olmesartan, eprosartan ( eprosartan) and azilsartan. In some embodiments, the additional therapeutic agent is an angiotensin receptor-neprilysin inhibitor (ARNi), e.g., Entresto®, sacubitril/valsartan; or an endothelin receptor body antagonists (ERA), for example, sitaxentan, ambrisentan, atrasentan, BQ-123, zibotentan, bosentan, horse Macitentan and tezosentan.

The invention also provides a kit comprising any dsRNA of the invention or any pharmaceutical composition of the invention, together with optional instructions for use. In one embodiment, the invention provides a kit for performing a method of inhibiting the expression of an AGT gene in a cell by contacting the cell with an amount of a double-stranded RNAi agent of the invention effective to inhibit the expression of AGT in the cell. The kit contains the RNAi agent and instructions for use, and, if desired, a method for administering the RNAi agent to an individual.

The present invention also provides a vial containing the dsRNA agent of the present invention or the pharmaceutical composition of the present invention. The present invention further provides a syringe containing the dsRNA agent of the present invention or the pharmaceutical composition of the present invention.

The invention further provides RNA-induced silencing complexes (RISC) comprising the antisense strand of any dsRNA agent of the invention.

In one embodiment, the RNAi agent is a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts" of each RNAi agent herein include, but are not limited to, sodium salts, calcium salts, lithium salts, potassium salts, ammonium salts, magnesium salts, and mixtures thereof. One of ordinary skill in the art will understand that when the RNAi agent is provided as a polycationic salt, each free acid group of the phosphodiester backbone and/or any other acidic modification (e.g., the 5'-terminal Phosphonate group) has one cation in it. For example, an oligonucleotide that is "n" nucleotides in length contains n-1 optionally modified phosphates, so an oligonucleotide that is 21 nt in length can be used as an oligonucleotide with up to 20 cations (e.g., 20 cations Sodium cation) salt is provided. Similarly, an RNAi agent with a sense strand of 21 nt in length and an antisense strand of 23 nt in length may be provided as a salt with up to 42 cations (eg, 42 sodium cations). In the above embodiments, when the RNAi agent also includes a 5'-terminal phosphate or a 5'-terminal vinyl phosphonate group, the RNAi agent can be as having up to 44 cations (e.g., 44 sodium cations) of salt provided.

The invention is further illustrated by the following detailed description.

The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript encoding the angiotensinogen (AGT) gene. The gene may be intracellular, for example, within an individual cell, such as a human. The use of these iRNAs initiates targeted degradation of the mRNA of the corresponding gene (AGT gene) in mammals.

The iRNA of the present invention is designed to target the human angiotensinogen (AGT) gene, including portions of the gene that are retained in AGT heterologues in other mammalian species. Without wishing to be bound by theory, it is believed that combinations or subcombinations of the above properties and specific sites or specific modifications in these iRNAs confer improved efficacy, stability, potency, durability and safety to the iRNAs of the invention.

Accordingly, the present invention provides methods of treating or preventing angiotensinogen (AGT)-related disorders, e.g., high blood pressure, hypertension, borderline hypertension, essential hypertension, secondary hypertension Hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, hypertonia pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy , chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, kidney disease, renal failure, systemic sclerosis disease, intrauterine growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes), and metabolic syndrome, using RNA-induced silencing complex (RISC)-mediated regulation of encoded vasculature iRNA composition of the cleaved RNA transcript of the protocinogen (AGT) gene.

iRNAs of the invention include RNA strands (antisense strands) containing regions up to about 30 nucleotides in length or less, for example, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23 or 21 to 22 A nucleotide length that is substantially complementary to at least a portion of the mRNA transcript of the AGT gene.

In some embodiments, the strands of one or more double-stranded RNAi agents of the invention are up to 66 nucleotides in length, e.g., 36 to 66, 26 to 36, 25 to 36, 31 to 60, 22 to 43 , 27 to 53 nucleotides in length, having a region of at least 19 contiguous nucleotides substantially complementary to at least a portion of the mRNA transcript of the AGT gene. In some embodiments, such iRNA agents with longer antisense strand lengths can, for example, include a second RNA strand (sense strand) of 20 to 60 nucleotides in length, wherein the sense strand and the The antisense strand forms a duplex of 18 to 30 consecutive nucleotides.

The use of iRNA of the present invention initiates targeted degradation of the mRNA of the corresponding gene (AGT gene) in mammals. Using in vitro assays, the inventors have demonstrated that iRNA targeting the AGT gene can efficiently mediate RNAi, resulting in significant inhibition of AGT gene expression. Accordingly, methods and compositions containing these iRNAs are useful for treating individuals suffering from AGT-related disorders, e.g., high blood pressure, hypertension, borderline hypertension, essential hypertension, Secondary hypertension, isolated systolic or diastolic hypertension, Pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, hypertension associated with low plasma renin activity or plasma renin concentration , intraocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, Diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, Systemic sclerosis, intrauterine fetal growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose tolerance Type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome.

Accordingly, the present invention provides methods and combination treatments for treating individuals with disorders that would benefit from inhibition or reduction of expression of the AGT gene, e.g., angiotensinogen I (AGT)-related disorders, e.g., high blood pressure pressure), hypertension (hypertension), borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, Refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart Disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricle Fibrosis, heart failure, myocardial infarction, angina, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic Steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes), and metabolic syndrome, using RNA-induced silencing complex (RISC)-mediated regulation of encoded vasculature iRNA composition of the cleaved RNA transcript of the protocinogen (AGT) gene.

The present invention also provides methods of preventing at least one symptom in an individual suffering from a disorder that would benefit from inhibition or reduction of AGT gene expression, for example, angiotensinogen (AGT)-related disorders, such as high blood pressure, hypertension (hypertension), borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, Paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, intraocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy , atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure , myocardial infarction, angina, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic steatohepatitis (NASH) ), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome.

The following detailed description discloses how to prepare and use iRNA-containing compositions to inhibit the expression of the AGT gene, as well as compositions, uses and methods for treating individuals who would benefit from inhibition and/or reduction of the expression of the AGT gene, e.g., susceptible to or individuals diagnosed with AGT-related disorders.

I.Definition

In order to make it easier to understand the present invention, some terms are first defined. Furthermore, it should be noted that whenever a value or range of a parameter is recited, that parameter's value or range is also interpolated between the intermediate values and ranges of the cited values to become part of this disclosure.

The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical objects of the article. As an example, "an element" refers to one element or a plurality of elements, for example, a plurality of elements.

The term "including" is used herein to mean the phrase "including, but not limited to," and may be used interchangeably with the phrase "including, but not limited to."

The term "or" is used herein to mean the phrase "and/or" and may be used interchangeably with the phrase "and/or" unless the context clearly dictates otherwise. For example, "equity shares or anti-indemnity shares" is understood to mean "equity shares or anti-indemnity shares or both indemnity shares and anti-indemnity shares".

As used herein, the term "about" means within the conventional tolerance range in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In some implementations, this means approximately ±10%. In some implementations, this means approximately ±5%. When about appears before a series of numbers or ranges, it should be understood that "about" can modify each number in the series or range.

The terms "at least", "no less than" or "or more" before a number or a series of numbers are understood to include the terms "at least" "Adjacent digits, and including all subsequent digits or integers, are clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides in a 21-nucleotide nucleic acid molecule" means that 19, 20, or 21 nucleotides have the specified property. when to When less appears before a series of numbers or ranges, it can be understood that "at least" can modify every number in the series or range.

As used herein, "no more than" or "or less" is understood to mean the value adjacent to the phrase and the logical lower value or integer to zero, logically from the context. . For example, a duplex with "no more than 2 nucleotides" overhangs has overhangs of 2, 1, and 0 nucleotides. When "no more than" appears before a series of numbers or ranges, it is understood that "no more than" modifies each number in the series or range. As used herein, ranges include upper and lower limits.

As used herein, a detection method may include determining that an analyte is present in an amount below the detection concentration of the method.

If the specified target site conflicts with the nucleotide sequence of the sense or antisense strand, the specified sequence takes precedence.

If a sequence conflicts with its designated position on transcription or other sequences, the nucleotide sequence described in the specification takes precedence.

As used herein, "angiotensinogen", used interchangeably with the term "AGT", refers to a well-known gene and polypeptide, also known in the art as Serpin Peptidase Inhibitor. , Clade A, Member 8; Alpha-1 anti-protease; anti-trypsin; SERPINA8; Angiotensin I; Serpin A8; Angiotensin II; Alpha-1 anti Protease angiotensinogen; antitrypsin; proangiotensinogen 2; ANHU; serpin; and cysteine protease inhibitor.

The sequence of the human AGT mRNA transcript can be found, for example, in GenBank accession numbers GI: 1813757520 (NM_000505.4; SEQ ID NO: 1; reverse complement, SEQ ID NO: 2) and NM_001384479.1 (SEQ ID NO: 3; reverse complement Complementary, SEQ ID NO: 4). The sequence of cynomolgus AGT mRNA can be found, for example, in GenBank accession number GI: 90075391 (NM_000029.1; SEQ ID NO: 5; reverse complement, SEQ ID NO: 6). The sequence of mouse AGT mRNA can be found, for example, in GenBank accession number GI: 113461997 (NM_007428.3; SEQ ID NO: 7; reverse complement, SEQ ID NO: 8). The sequence of rat AGT mRNA can be found, for example, in GenBank accession number GI: 51036672 (NM_134432.2; SEQ ID NO: 9; reverse complement, SEQ ID NO: 10). The sequence of Macaca mulatta AGT mRNA can be found, for example, in GenBank XM_015126038 (SEQ ID NO: 11; reverse complement, SEQ ID NO: 12).

Other examples of AGT mRNA sequences are readily available through public databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

Further information about AGT can be found at, for example, www.ncbi.nlm.nih.gov/gene/? term=AGT.

The entire contents of each of the foregoing GenBank accession numbers and gene database numbers are incorporated herein by reference as of the date of filing of this application.

_ref.cgi？geneId=183上的dbSNP數據庫中找到。AGT基因內的序列變化的非限制性實施例包括，例如，美國專利號5,589,584中描述的那些，其全部內容藉由引用併入本文。例如， AGT基因內的序列變異可能包括位置-532(相對於轉錄起始位點)的C→T；在位置-386的G→A；在位置-218的G→A；在位置-18的C→T；在位置-6及-10的G→A和A→C；在位置+10的C→T(未轉譯)；在位置+521的C→T(T174M)；在位置+597的T→C(P199P)；位置+704的T→C(M235T；另見，例如，參考SNP(refSNP)聚類報告(Cluster Report)：rs699，可在www.ncbi.nlm.nih.gov/SNP獲得)；在位置+743的A→G(Y248C)；在位置+813的C→T(N271N)；在位置+1017的G→A(L339L)；在位置+1075的C→A(L359M)；和/或位置+1162的G→A(V388M)。 As used herein, the term "AGT" also refers to naturally occurring DNA sequence variations of the AGT gene, such as single nucleotide polymorphisms (SNPs) in the AGT gene. Exemplary SNPs are available at www.ncbi.nlm.nih.gov/projects/SNP/snp

_ref.cgi? Found in the dbSNP database on geneId=183. Non-limiting examples of sequence changes within the AGT gene include, for example, those described in U.S. Patent No. 5,589,584, the entire contents of which are incorporated herein by reference. For example, sequence variations within the AGT gene might include C→T at position -532 (relative to the transcription start site); G→A at position -386; G→A at position -218; C→T; G→A and A→C at positions -6 and -10; C→T at position +10 (untranslated); C→T at position +521 (T174M); at position +597 T→C (P199P); T→C (M235T) at position +704; see also, e.g., Reference SNP (refSNP) Cluster Report: rs699, available at www.ncbi.nlm.nih.gov/SNP Obtain); A→G at position +743 (Y248C); C→T at position +813 (N271N); G→A at position +1017 (L339L); C→A at position +1075 (L359M) ; and/or G→A(V388M) at position +1162.

As used herein, "target sequence" refers to the contiguous portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the AGT gene, including the mRNA as a product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during the transcription of the AGT gene.

The target sequence may be about 19 to 36 nucleotides in length, such as about 19 to 30 nucleotides in length. For example, the target sequence may be about 19 to 30 nucleotides, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, A length of 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides. In some embodiments, the target sequence is 19 to 23 nucleotides in length, optionally 21 to 23 nucleotides in length. Ranges and lengths intermediate to the ranges and lengths recited above are also considered part of the present disclosure.

As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides described by a sequence referred to using standard nucleotide nomenclature.

"G", "C", "A", "T" and "U" respectively represent nucleotides based on guanine, cytosine, adenine, thymidine and uracil bases. However, it should be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, as described in further detail below, or to substituted moieties (see, e.g., Table 1). It will be apparent to those of ordinary skill that guanine, cytosine, adenine and uracil may be substituted by other moieties without significantly altering the base pairing properties of oligonucleotides containing nucleotides bearing such substituted moieties . For example, but not limited to, a nucleotide containing inosine as its base may base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, uracil, guanine, or adenine-containing nucleotides may be substituted with, for example, inosine-containing nucleotides in the nucleotide sequence of the dsRNA featured in the present disclosure. In another embodiment, adenine and cytosine at any position in the oligonucleotide can be replaced by guanine and uracil, respectively, thereby forming G-U Wobble base pairing with the target mRNA respectively. Sequences containing such substituted moieties are suitable for use in the compositions and methods of the present invention.

As used interchangeably herein, the terms "iRNA", "RNAi agent", "iRNA agent" and "RNA interference agent" refer to an RNA containing the term as defined herein, and which acts through the RNA-induced silencing complex (RISC) pathway and mediates targeted cleavage of RNA transcription. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits the expression of the AGT gene in cells, e.g., liver cells in an individual, such as a mammalian individual.

In one embodiment, the RNAi agents of the invention comprise single-stranded RNA that interacts with a target RNA sequence (eg, an AGT target mRNA sequence) to direct cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is cleaved into siRNA by a type III endonuclease called Dicer (Sharp et al. (2001) genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dRNA into short interfering RNA of 19 to 23 base pairs, which has a characteristic two-base 3' overhang (Bernstein, et al. , (2001) Nature 409: 363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, initiating the complementary antisense strand to lead to target recognition (Nykanen, et al. , (2001) Cell 107:309). After binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al. , (2001) genes Dev. 15:188). Therefore, one aspect of the present disclosure relates to intracellularly produced single-stranded RNA (siRNA) that promotes the formation of RISC complexes to affect silencing of a target gene, namely the AGT gene. Therefore, the term "siRNA" is also used herein to refer to iRNA as described above.

In some embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi), which is introduced into a cell or organism to inhibit target mRNA. This single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2 and then cleaves the target mRNA. Single-stranded siRNA is usually 15 to 30 nucleotides in length and chemically modified. The design and testing of single-stranded siRNA is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each patent being incorporated herein by reference. Any antisense nucleotide sequence described herein can be used as a single-stranded siRNA as described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In some embodiments, "iRNA" used in the compositions, uses, and methods of the present disclosure is double-stranded RNA, and is referred to herein as "double-stranded RNA agent" or "double-stranded RNA (dsRNA) molecule" , "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules, containing a duplex structure containing two antiparallel and substantially complementary nucleic acid strands, termed "containing" relative to the target RNA (i.e., the AGT gene). "sense" and "antisense" directions, that is, the AGT gene. In some embodiments of the present disclosure, the double-stranded RNA (dsRNA) triggers the degradation of target RNA, eg, mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

Typically, the majority of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides nucleotides or modified nucleotides. In addition, as used in this specification, "iRNA" may include ribonucleotides with chemical modifications; iRNA may include substantial modifications on multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleoside containing a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, independently or in any combination thereof acid. Thus, the term modified nucleotide includes substitutions, additions or removals of, for example, functional groups or atoms, to internucleoside linkages, sugar moieties or nucleobases. Modifications suitable for use in the agents of the present disclosure include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modification as used in siRNA-type molecules is included within "iRNA" or "RNAi agent."

In some embodiments of the present disclosure, deoxy-nucleotides, if included in the RNAi agent, may be considered to constitute modified nucleotides.

The duplex region of any length that allows specific degradation of the desired target RNA by the RISC pathway can be in the range of about 19 to 36 base pairs in length, for example, about 19 to 30 base pairs in length, For example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35 or 36 base pairs in length, such as about 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21 , 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length. In some embodiments, the duplex region is 19 to 21 base pairs in length, for example, 21 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

The two strands forming a duplex structure may be different parts of a larger RNA molecule, or they may be separate RNA molecules. If the two strands are part of a larger molecule, they are connected by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the other strand forming a duplex structure , the connected RNA strands are called "hairpin loops." The hairpin loop may contain at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can contain 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can contain 4 to 10 unpaired nucleotides. In some embodiments, the hairpin loop can range from 4 to 8 nucleotides.

If the two substantially complementary strands in the dsRNA consist of separate RNA molecules, these molecules need not be, but can be, covalently linked. If two strands are covalently linked by any means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the other strand forming a duplex structure, the linked structure It's called a "linker". The RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the duplex. In addition to duplex structures, RNAi can also contain one or more nucleotide overhangs. In one embodiment of the RNAi agent, the at least one strand includes a 3' overhang of at least 1 nucleotide. In another embodiment, the at least one strand includes a 3' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent includes a 5' overhang of at least 1 nucleotide. In some embodiments, at least one strand includes a 5' overhang comprising at least 2 nucleotides, e.g., 2, 3, 4, 5, 16, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In some embodiments, the iRNA agent of the present invention is a dsRNA, each of which contains 19 to 23 nucleotides, which interacts with the target RNA sequence to guide the cleavage of the target RNA, for example, the AGT gene.

In some embodiments, iRNAs of the invention are 24 to 30 nucleotide dsRNAs that interact with a target RNA sequence to direct cleavage of the target RNA, for example, an AGT target mRNA sequence.

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of a double-stranded iRNA. For example, when dsRNA Nucleotide overhangs exist when the 3'-end of one strand extends beyond the 5'-end of the other strand, or vice versa. The dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. Nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides. The prominence can be on shares, anti-sense shares, or any combination thereof. In addition, overhanging nucleotides can be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the dsRNA.

In one embodiment, the antisense strand of the dsRNA protrudes from the 3'-end or the 5'-end by 1 to 10 nucleotides, for example, 1, 2, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In one embodiment, the sense strand of the dsRNA protrudes from the 3'-end or the 5'-end by 1 to 10 nucleotides, for example, 1, 2, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with nucleoside phosphorothioates.

In some embodiments, the antisense strand of the dsRNA protrudes at the 3'-end or the 5'-end by 1 to 10 nucleotides, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10. For example, 1, 2, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In one embodiment, the sense strand of the dsRNA protrudes from the 3'-end or the 5'-end by 1 to 10 nucleotides, for example, 1, 2, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with nucleoside phosphorothioates.

In some embodiments, the antisense strand of the dsRNA protrudes from the 3' end or the 5' end with 1 to 10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In some embodiments, overhangs on the sense strand or antisense strand, or both, may include extended lengths of more than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides , 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleosides acid length. In some embodiments, the extended overhang is located on the sense strand of the duplex. In some embodiments, the extended overhang is present at the 3' end of the sense strand of the duplex. In some embodiments, the extended overhang is present at the 5' end of the sense strand of the duplex. In certain embodiments, the extended overhang is located on the antisense strand of the duplex. In some embodiments, the extended overhang is present at the 3' end of the antisense strand of the duplex. In some embodiments, the extended overhang is present at the 5' end of the duplex that contains the antisense strand. In some embodiments, one or more nucleotides in the expanded overhang are replaced with nucleoside phosphorothioates. In some embodiments, the protrusions include self-complementary portions that enable the protrusions to form hairpin structures that are stable under physiological conditions.

"Blunt" or "blunt end" means that there are no unpaired nucleotides at the end of the double-stranded RNA agent, that is, there are no nucleotide overhangs. "Blunt-ended" double-stranded RNA agents are double-stranded throughout their entire length, that is, there are no nucleotide overhangs at either end of the molecule. RNAi agents of the present disclosure include RNAi agents that do not have a nucleotide overhang at one end (ie, an agent has an overhang and a blunt end) or that do not have a nucleotide overhang at either end. In most cases, the molecule is double-stranded throughout its length.

The term "antisense strand" or "guide strand" refers to the strand of iRNA, e.g., dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., AGT mRNA.

As used herein, the term "region of complementarity" refers to a region on an antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an AGT nucleotide sequence, as defined herein. If the complementary region is not completely complementary to the target sequence, the mismatch may be in the internal or terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal region, e.g., within 5, 4, or 3 nucleotides of the 5'- or 3'-end of the iRNA. In some implementation aspects, the present disclosure Double-stranded RNA agents include nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present disclosure includes no more than 4 mismatches with the target mRNA. For example, the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. mismatch. In some embodiments, the double-stranded RNA agent of the antisense strand of the present disclosure includes no more than 4 mismatches with the sense strand. For example, the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. stock mismatch. In some embodiments, double-stranded RNA agents of the present disclosure include nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present disclosure includes no more than 4 mismatches with the antisense strand. For example, the sense strand includes 4, 3, 2, 1, or 0 mismatches with the antisense strand. stock mismatch. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, or 3 nucleotides from the 3'-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3'-terminal nucleotide of the iRNA agent. In some implementations, the mismatch is not in the seed region.

Thus, an RNAi agent as described herein may contain one or more mismatches to the target sequence. In one embodiment, an RNAi agent as described herein contains no more than 3 mismatches (ie, 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In some embodiments, if the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch can optionally be limited to the last 5 nucleotides from the 5'- or 3'-end of the complementary region. Inside the acid. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand complementary to the region of the AGT gene will typically not contain any mismatches within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting AGT genes. Because of performance. It is important to consider the effects of RNAi agents that are mismatched to the inhibitory performance of the AGT gene, particularly if a specific complementary region in the AGT gene is known to have polymorphic sequence variation in the population.

As used herein, the term "sense strand" or "passenger strand" refers to an iRNA strand that contains a region that is substantially complementary to a region of the antisense strand as defined herein.

As used herein, "substantially all of the nucleotides are modified" is modified to a large extent, but not entirely, and may include no more than 5, 4, 3, 2 or 1 unmodified nucleotide.

As used herein, the term "cleavage region" refers to the region immediately adjacent to the cleavage site. The cleavage site is the point on the target where cleavage occurs. In some embodiments, the cleavage region includes three bases at either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage region includes two bases at either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage site occurs specifically at a site that binds nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

As used herein, unless otherwise stated, when the term "complementary" is used to describe a first nucleotide sequence that is related to a second nucleotide sequence, it refers to an oligonucleotide that includes the first nucleotide sequence. The ability of an acid or polynucleotide, under certain conditions using an oligonucleotide or polynucleotide containing a second nucleotide sequence, to hybridize and form a duplex structure, as will be understood by one of ordinary skill. Such conditions can be, for example, stringent conditions, where stringent conditions can be: 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50°C or 70°C for 12 to 16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may be applied, such as physiologically relevant conditions that may be encountered in vivo. One with ordinary knowledge will be able to determine the final application of the hybrid nucleotides. Test the complementarity of two sequences to determine the most suitable set of conditions.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include an oligonucleotide or polynucleotide containing a first nucleotide sequence and a second core containing the entire length of one or both nucleotide sequences. Base pairing of oligonucleotides or polynucleotides. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" with respect to a second sequence, the two sequences may be completely complementary, or they may form one or more, but generally not More than 5, 4, 3 or 2 mismatched bases, in hybridizing a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most relevant to its end application, e.g., in vitro or in vivo Inhibit gene expression. However, when two oligonucleotides are designed to form one or more single-stranded overhangs when hybridized, such overhangs should not cause visual mismatches in determining complementarity. For example, a dsRNA containing one oligonucleotide that is 21 nucleotides in length and another oligonucleotide that is 23 nucleotides in length, where the longer oligonucleotide contains the same oligonucleotide as the shorter oligonucleotide. A sequence of 21 nucleotides that is completely complementary to an acid may still be referred to as "fully complementary" for the purposes described herein.

As used herein, "complementary" sequences may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed of non-natural and modified nucleotides, so long as the above with respect to their ability to hybridize Requirements are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogsteen base pairs.

"Complementary", "fully complementary" and "substantially complementary" as used herein may be used with respect to the sense strand and the antisense strand of dsRNA or between two oligonucleotides or Base matching between polynucleotides, such as the antisense strand and target sequence of a double-stranded RNA agent, is understood from the context in which they are used.

As used herein, a polynucleotide that is "substantially complementary to at least part of" a message RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding the AGT gene). complementary polynucleotides. For example, a polynucleotide is complementary to at least a portion of an AGT mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding the AGT gene.

Therefore, in some embodiments, the antisense polynucleotides disclosed herein are completely complementary to the target AGT sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target AGT sequence and comprise SEQ ID NO: 1, 3, 5, 7, 9 or 11, or SEQ ID NO. NO: The parallel region of the nucleotide sequence of any of the fragments 1, 3, 5, 7, 9 or 11 is at least 80% complementary to the continuous nucleotide sequence, such as about 85%, about 90%, about 91% , about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target AGT sequence and include any sense nucleotide sequence consistent with any one of Tables 2 to 7 or Table 2 over its entire length. Any segment of any one of the sense nucleotide sequences to 7 is at least about 80% complementary to the contiguous nucleotide sequence, such as about 85%, about 90%, about 91%, about 92%, about 93%, About 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% complementary.

In one embodiment, the RNAi agent of the present disclosure includes an antisense polynucleotide that is substantially complementary to an antisense polynucleotide that is identical to the target AGT sequence, and wherein the sense polynucleotide The acid comprises a contiguous nucleotide sequence that corresponds over its entire length to the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 or to SEQ ID NO: 2, 4, 6, 8, 10 or 12 The equivalent regions of any segment are at least about 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, About 98%, about 99% or 100% complementary.

In some embodiments, the iRNA of the present disclosure is substantially complementary to an antisense polynucleotide, which in turn is complementary to a target AGT sequence, and wherein the sense polynucleotide includes contiguous A nucleotide sequence that is at least about 80% identical in its entire length to any sense nucleotide sequence in any one of Tables 2 to 7 or to a fragment of any sense nucleotide sequence in any one of Tables 2 to 7 % complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100 % complementary.

Typically, "iRNA" includes chemically modified ribonucleotides. Such modifications may include all types of modifications described herein or known in the art. For the purposes of this specification and claims, any such modification is included in "iRNA", as used in dsRNA molecules.

In some embodiments of the present disclosure, deoxynucleotides may be considered to constitute modified nucleotides if present in the RNAi agent.

In one aspect of the present disclosure, agents used in the methods and compositions of the present disclosure are single-stranded antisense oligonucleotide molecules that inhibit target mRNA through an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. This single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by base pairing with the mRNA and physically blocking the translation machinery, see Dias, N. et al. , (2002) Mol Cancer Ther 1: 347-355 . The single-stranded antisense oligonucleotide molecule can be about 14 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule can comprise a sequence of at least about 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any of the antisense sequences described herein .

As used herein, the phrase "contacting a cell with an iRNA", such as dsRNA, includes contacting the cell by any possible means. Contacting the cell with the iRNA includes contacting the cell with the iRNA in vitro or contacting the cell with the iRNA in vivo. This contact can be directed or indirect. Thus, for example, the iRNA can be brought into physical contact with the cell by the individual performing the method, or the iRNA can be placed in an environment that permits or results in its subsequent contact with the cell.

Contacting cells in vitro can be accomplished, for example, by incubating the cells with iRNA. Contact of cells in vivo can be achieved, for example, by injecting iRNA into or near the tissue where the cells are located or by injecting iRNA into another area, e.g., the bloodstream or subcutaneous space, so that the agent will then reach the location of the cells to be contacted. organization. For example, the iRNA can comprise or be coupled to a ligand, eg, GalNAc, which directs the iRNA to a site of interest, eg, the liver. Combinations of in vitro and in vivo contact methods are also possible. For example, cells can also be exposed to iRNA outside the body and then transplanted into an individual.

In some embodiments, contacting the iRNA with the cell includes "introducing" or "delivering the iRNA into the cell" by promoting or affecting cellular uptake or uptake. Uptake or uptake of iRNA can occur by independent diffusion or active cellular processes, or by auxiliary agents or devices. Introducing iRNA into cells can In vitro or in vivo. For example, for in vivo introduction, iRNA can be injected into the tissue site or administered systemically. Introduction of cells in vitro includes methods known in the art, such as electroporation and lipofection. Further methods are described below or are known in the art.

The term "lipid nanoparticle" or "LNP" refers to vesicles in which a lipid layer encapsulates a pharmaceutically active molecule (eg, a nucleic acid molecule), such as iRNA, or a plasmid from which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

As used herein, "subject" refers to an animal, such as a mammal, including primates (e.g., humans, non-human primates (e.g., monkeys), and chimpanzees), non-primates (e.g., cattle) , pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat or mouse), or a bird expressing the target gene endogenously or heterologously. In one embodiment, the system is a human being, such as a person who is being treated or evaluated for a disease or disorder that would benefit from reduced AGT performance; a person who is likely to benefit from a disease or disorder that would benefit from reduced AGT performance; a person who has a disease or disorder that would benefit from reduced AGT performance; A person with a disease or disorder that would benefit from a reduction in performance; or a person who is being treated for a disease or disorder that would benefit from a reduction in AGT performance as described herein. Diagnostic criteria for AGT-related disorders, such as hypertension, are provided below. In some implementations, the system is female. In other implementations, the system is male. In some embodiments, the system is part of a population susceptible to salt sensitivity, for example, black people or the elderly (>65 years old). In some embodiments, the system is overweight or obese, for example, an individual suffers from central obesity. In some implementations, the system is sedentary. In some implementations, the system is configured. In another implementation, the system is a child entity.

As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result, such as reducing the signs or symptoms of at least one AGT-related disorder in an individual. Treatment also includes reducing one or more signs or symptoms associated with unwanted AGT manifestations; reducing the degree of unwanted AGT activation or stabilization; ameliorating or alleviating unwanted AGT activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with unwanted AGT, e.g., angiotensin II type 1 receptor activation (AT1R) (e.g., hypertension, chronic kidney disease, stroke, myocardial infarction, heart failure , aneurysm, peripheral arterial disease, cardiac disease, increased oxidative stress, increased superoxide formation, inflammation, vasoconstriction, sodium and water retention, potassium and magnesium loss, renin inhibition, myocyte and smooth muscle hypertrophy, increased collagen synthesis , stimulation of vascular, myocardial and renal fibrosis, increased cardiac contraction rate and force, changes in heart rate, increased arrhythmia, stimulation of plasminogen activator inhibitor 1 (PAI1), activation of the sympathetic nervous system and increased secretion of endothelin) , pregnancy-related symptoms of hypertension (such as preeclampsia and eclampsia), including, but not limited to, intrauterine fetal growth retardation (IUGR) or fetal growth retardation, symptoms associated with malignant hypertension, and hyperaldosteronism. Symptoms; reduction in unwanted AT1R activation; stabilization (i.e., no worsening) of chronic AT1R activation; improvement or reduction in unwanted AT1R activation (e.g., hypertension, chronic kidney disease, stroke, myocardial infarction, heart failure, arterial tumors, peripheral arterial disease, cardiac disease, increased oxidative stress, e.g., increased superoxide formation, inflammation, vasoconstriction, sodium and water retention, potassium and magnesium losses, renin inhibition, myocyte and smooth muscle hypertrophy, increased collagen synthesis, Stimulation of vascular, myocardial and renal fibrosis, increased speed and force of cardiac contractions, changes in heart rate, e.g. increased arrhythmias, stimulation of plasminogen activator inhibitor 1 (PAI1), activation of the sympathetic nervous system and endothelin secretion), whether detectable or undetectable. AGT-related diseases may also include obesity, hepatic steatosis/fatty liver disease, such as non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD), glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome. In some embodiments, hypertension includes hypertension associated with low plasma renin activity or plasma renin concentration. "Treatment" can also mean prolonging survival compared to expected survival without treatment.

The term "lower" in the context of AGT concentrations in an individual or disease marker or symptom refers to a statistically significant decrease in such concentrations. The reduction may be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% %, 85%, 90%, 95% or more. In some implementations, the reduction is at least 20%. In some embodiments, the reduction is at least a 50% reduction in disease marker, eg, protein, protein or gene concentration. In an individual, "lower" in the context of AGT concentrations refers to a decrease to a concentration that is within the normal range acceptable for an individual without such disorder. In some embodiments, "lower" refers to a decrease in the difference between the concentration that is a marker or symptom of a disease in an individual and the concentration that the individual receives within the normal range. The term "lower" may also be used in connection with normalization of symptoms of a disease or condition, i.e., a reduction in the concentration in an individual with an AGT-related disorder compared with the concentration in a normal individual without the disease or condition. difference. AGT-related disorders. As used herein, if a disease is associated with an elevated value of a symptom, then "normal" is considered the upper limit of normal. If the disease is associated with a reduced value of the symptom, then "normal" is considered the lower limit of normal.

As used herein, "prevention" or "preventing", when used to refer to a disease or disorder, e.g., in an individual susceptible to an AGT-related disorder, would benefit from a reduction in AGT gene expression or AGT Protein production, for example, due to aging, genetic factors, hormonal changes, diet and sedentary lifestyle. In certain embodiments, the disease or condition is, e.g. Symptoms of unwanted AT1R activation, such as hypertension, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysm, peripheral arterial disease, heart disease, increased oxidative stress, such as increased superoxide formation, inflammation, vasoconstriction , sodium and water retention, potassium and magnesium losses, renin inhibition, myocyte and smooth muscle hypertrophy, increased collagen synthesis, stimulation of vascular, myocardial and renal fibrosis, increased cardiac contraction rate and force, heart rate changes such as increased arrhythmias, fiber Stimulation of lysinogen activator inhibitor 1 (PAI1), activation of the sympathetic nervous system, and increased endothelin secretion. AGT-related diseases also include obesity, hepatic steatosis/fatty liver disease such as non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD), glucose intolerance, and type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome. In certain embodiments, hypertension includes hypertension associated with low plasma renin activity or plasma renin concentration. For example, when an individual with one or more risk factors for hypertension does not have hypertension or develops hypertension of a lower severity relative to a population with the same risk factors who do not receive treatment as described herein, e.g. The likelihood of high blood pressure is reduced. Not having an AGT-related disease, such as high blood pressure, or delaying the onset of high blood pressure for months or years is considered effective prevention. Prevention may require administration of more than one dose of iRNA agent.

As used herein, the term "angiotensinogen-associated disease" or "AGT-associated disease" is caused by or related to the renin-angiotensin-aldosterone system (RAAS) Disease or disorder, or symptoms or worsening of a disease or disorder that is responsive to RAAS inactivation. The term "provasotocin-related diseases" includes diseases, disorders or conditions that would benefit from reduced expression of AGT, and these diseases are commonly associated with hypertension. Non-limiting examples of provasotocin-related diseases include hypertension, e.g., borderline hypertension (also known as prehypertension), essential hypertension (also known as essential or idiopathic hypertension) , secondary hypertension (also called non-essential hypertension), isolated systolic blood pressure or diastolic blood pressure Diastolic hypertension, pregnancy-related hypertension (eg, preeclampsia, eclampsia, and postpartum preeclampsia), diabetic hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension (also known as Renal hypertension), Goldblatt hypertension, intraocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis sclerosis, arteriosclerosis, vascular disease (including peripheral vascular disease), diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, Ventricular fibrosis, sleep apnea, heart failure (e.g., left ventricular systolic dysfunction), myocardial infarction, angina, stroke, renal disease (e.g., chronic kidney disease or diabetic nephropathy, as appropriate in the setting of pregnancy), renal failure (e.g., chronic renal failure) and systemic sclerosis (e.g., sclerosing kidney crisis). In some embodiments, AGT-related disorders also include intrauterine growth retardation (IUGR) or fetal growth retardation. In some implementations, AGT-related disorders also include obesity, hepatic steatosis/fatty liver disease, e.g., non-alcoholic fatty liver disease (NASH) and non-alcoholic fatty liver disease (NAFLD), glucose intolerance, type 2 Diabetes (non-insulin-dependent diabetes mellitus)) and metabolic syndrome. In some embodiments, hypertension includes hypertension associated with low plasma renin activity or plasma renin concentration.

Thresholds of hypertension and stages of hypertension are discussed in detail below.

In one implementation, the pro-angiotocin-related disease is essential hypertension. "Primary hypertension" is the result of environmental or genetic causes (for example, without an obvious underlying medical cause).

In one implementation, the pro-angiotocin-related disease is secondary hypertension. "Secondary hypertension" has an identifiable underlying disorder and may have multiple etiologies, including renal, vascular and endocrine causes, for example, renal parenchymal disease (eg, multiple cystic kidney disease, glomerular or interstitial disease), renovascular disease (eg, renal artery stenosis, fibromuscular dysplasia), endocrine disorders (eg, adrenocorticoid or mineralocorticoid excess, pheochromocytoma, hyperthyroidism or hypothyroidism, growth hormone excess, hyperparathyroidism), coarctation of the aorta, or use of oral contraceptives.

In one embodiment, the provasodilator-related disease is pregnancy-related hypertension, for example, chronic hypertension of pregnancy, gestational hypertension, preeclampsia, eclampsia, preeclampsia superimposed on chronic hypertension, HELLP Syndrome, and gestational hypertension (also known as transient hypertension of pregnancy, chronic hypertension detected in the second half of pregnancy, and pregnancy-induced hypertension (PIH)). Diagnostic criteria for pregnancy-related hypertension are provided below.

In one implementation, the pro-angiotocin-related disease is resistant hypertension. "Resistant hypertension" refers to an individual whose blood pressure remains above target (e.g., systolic blood pressure above 130 mm Hg) despite concurrent use of three different classes of antihypertensive agents, one of which is a thiazide diuretic or diastolic blood pressure higher than 90mm Hg). Individuals who control their blood pressure with four or more medications are also considered to have resistant hypertension.

As used herein, a "therapeutically effective amount" is intended to include an amount of RNAi sufficient to effect treatment of disease (e.g., by reducing, ameliorating, or maintaining existing disease or disease) when administered to an individual with an AGT-related disorder. one or more symptoms of a disease). A "therapeutically effective amount" may vary depending on the RNAi agent, the manner in which the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and Other personal characteristics of the individual to be treated.

As used herein, "prophylactically effective amount" is intended to include an amount sufficient to prevent or ameliorate AGT-related disorders when administered to an individual with an AGT-related disorder. An amount of an RNAi agent that ameliorates a disorder or symptoms of one or more disorders. Ameliorating the disease includes slowing the progression of the disease or reducing the severity of later development of the disease. A "prophylactically effective amount" may vary depending on the RNAi agent, the manner in which the agent is administered, the degree of disease risk and medical history, age, weight, family history, genetic makeup, the type of prior or concomitant therapy (if any), and the treatment to be treated Other personal characteristics of each individual.

A "therapeutically-effective amount" or a "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any treatment. The iRNA used in the methods of the present disclosure can be administered in sufficient amounts to produce a reasonable benefit/risk ratio suitable for such treatment.

As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials (including salts), compositions, which are suitable, within the scope of sound medical judgment, for use in contact with tissue of human and animal subjects. or dosage form, without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase "pharmaceutically-acceptable carrier" refers to a pharmaceutically acceptable material composition or carrier, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricants, magnesium talc, calcium or zinc stearate, or stearic acid) or solvent-embedded materials containing a subject compound that carries or transports the subject compound from one organ or part of the body to another a part of. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject treated. Such vectors are known in the art. Pharmaceutically acceptable carriers include those administered by injection.

As used herein, the term "sample" includes similar body fluids, cells, or tissues isolated from an individual, as well as collections of body fluids, cells, or tissues in an individual. Examples of biological fluids include blood, serum and serum, plasma, cerebrospinal fluid, eye fluid, lymph fluid, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or local areas. For example, the sample may be from specific organs, parts of organs, or fluids or cells within those organs. In some embodiments, the sample may be obtained from the liver (eg, a whole liver or a partial liver segment or a subset of cell types in the liver, eg, hepatocytes). In some implementations, "sample derived from a subject" refers to urine obtained from a subject. "Sample derived from a subject" may mean serum or plasma derived from the blood or blood of an individual.

II. iRNAs of the present invention

The present invention provides iRNAs that inhibit the expression of AGT genes. In some embodiments, the iRNA includes a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting AGT gene expression in cells, such as cells within an individual, such as a mammal, such as a human susceptible to AGT-related disorders such as hypertension. The dsRNAi agent includes an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during expression of the AGT gene. The complementary region is approximately 19-30 nucleotides in length (e.g., approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 or 19 nucleotides in length) .

Upon contact with a cell expressing the AGT gene, the iRNA inhibits the expression of the AGT gene (e.g., human, primate, non-primate, or rat AGT gene) by at least about 50%, as measured, for example, by, PCR or branched DNA (bDNA) based methods, or by protein based methods, such as by immunofluorescence analysis, using, for example, Western blotting or flow cytometry techniques. In some embodiments, performance is inhibited by The qPCR method provided is determined by the appropriate biological cell line in which to provide, for example, a 10 nM concentration of siRNA. In some embodiments, inhibition of expression in vivo is determined by knocking down the human gene in a rodent expressing the human gene (e.g., a mouse expressing a human target gene or an AAV-infected mouse), e.g., a mouse or AAV-infected mice expressing the human target gene, for example, when administered as a single dose, for example, at 3 mg/kg at the nadir of RNA expression.

dsRNA consists of two complementary RNA strands that are complementary and hybridize to form a duplex structure in the case of dsRNA. One strand of dsRNA (the antisense strand) contains a complementary region that is substantially, and usually completely, complementary to the target sequence. The target sequence can be derived from the mRNA sequence formed during expression of the AGT gene. The other strand (the sense strand) contains regions complementary to the antisense strand, allowing the two strands to combine under appropriate circumstances to form a duplex structure. As described elsewhere herein and known in the art, the complementary sequence of the dsRNA can also be contained in self-complementary regions as a single nucleic acid molecule rather than tethered to a separate oligonucleotide.

Typically, the double-stranded structure is 15 to 30 base pairs in length, for example, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23 or 21 to 22 base pairs. In some embodiments, the double-stranded structure is 18 to 25 base pairs in length, for example, 18 to 25, 18 to 24, 18 to 25 base pairs. 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24 or 24 to 25 bases Pair length, for example, 19 to 21 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

Similarly, regions complementary to the target sequence are 15 to 30 nucleotides in length, e.g., 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23 , 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23 , 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23 or 21 to 22 nucleotides in length, such as 19 to 23 Nucleotide length or 21 to 23 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

In some embodiments, the double-stranded structure is 19 to 30 base pairs in length. Similarly, regions complementary to target sequences are 19 to 30 nucleotides in length.

In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally speaking, the length of the dsRNA is sufficient to serve as a target for the Dicer enzyme. For example, it is well known in the art that more than about 21 to 23 A dRNA with a length of 3 nucleotides can serve as a substrate for Dicer. As one of ordinary skill will also recognize, the region of the RNA targeted for cleavage is typically part of a larger RNA molecule, typically an mRNA molecule. If relevant, a "part" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

One of ordinary skill in the art will also recognize that the major functional portion of the duplex region dsRNA, e.g., the duplex region of about 19 to about 30 base pairs, e.g., about 19 to 30, 19 to 29 , 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25 , 21 to 24, 21 to 23 or 21 to 22 base pairs. Thus, in one embodiment, the desired RNA targeting for cleavage has greater than 30 bases to the extent that it is processed into a functional duplex, such as 15 to 30 base pairs. The RNA molecule or RNA molecule complex of the duplex region is dsRNA. Accordingly, one of ordinary skill will recognize that in one embodiment, the miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, iRNA agents for targeting AGT gene expression are not produced in target cells by cleavage of larger dsRNA.

The dsRNA as described herein may further comprise one or more single-stranded nucleotide overhangs, such as 1 to 4, 2 to 4, 1 to 3, 2 to 3, 1, 2, 3 or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have better inhibitory properties relative to their blunt-ended counterparts. Nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs, including Oxynucleotides/nucleosides. The prominence can be on the sense stock, the antisense stock, or any combination thereof. In addition, overhanging nucleotides may be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the dsRNA.

dsRNA can be synthesized by standard methods known in the art. Double-stranded RNAi compounds of the invention can be prepared using a two-step procedure. First, each strand of the double-stranded RNA molecule is prepared individually. Then, glue the component strands. Solution phase or solid phase organic synthesis or both can be used to prepare single strands of siRNA compounds. Organic synthesis offers the advantage that oligonucleotide strands containing non-natural or modified nucleotides can be readily prepared. Similarly, single-stranded oligonucleotides of the invention can be prepared using solution phase or solid phase organic synthesis, or both.

In one aspect, the dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense stock is selected from the group of any sequence provided in Tables 2 to 7, and the antisense stock corresponding to the sense stock is selected from the group of any sequence provided in Tables 2 to 7. In this regard, one of the two sequences is complementary to the other of the two sequences, one of which is substantially complementary to the mRNA sequence produced in the expression of the AGT gene. Therefore, in this aspect, the dsRNA will comprise two oligonucleotides, one of which is the sense strand described in any one of Tables 2 to 7 and the second oligonucleotide is the one described in Tables 2 to 7 Any of the inverse shares corresponding to the inverse shares. .

In some embodiments, substantially complementary sequences of the dsRNA are contained in separate oligonucleotides. In other embodiments, substantially complementary sequences of the dsRNA are contained in a single oligonucleotide.

It will be understood that, although the sequences in, for example, Table 2 are not described as modified or splice sequences, the RNA of the iRNA of the invention, for example, the dsRNA of the invention, may comprise the unmodified, unspliced sequences listed in any of Tables 2 to 7. , or modify or incorporate sequences different from those described therein. In other words, as described herein, the present disclosure encompasses the dsRNAs of Tables 2 to 7, whether unmodified, unligated, modified, or conjugated.

It is well known to those of ordinary skill that dsRNAs containing duplex structures of about 20 to 23 base pairs, for example, 21 base pairs are favored because they are particularly effective in inducing RNA interference (Elbashir et al. , EMBO 2001 ,20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226) . In the above embodiments, due to the properties of the oligonucleotide sequences provided in any of Tables 2 to 7. The dsRNA described herein can include at least one strand of at least 21 nucleotides in length. It is reasonable to expect that shorter duplexes with any one of the sequences in Tables 2 to 7 minus only a few nucleotides on one or both ends could be equally effective compared to the dsRNA described above. Thus, dsRNAs with a sequence of at least 19, 20, or more contiguous nucleotides derived from the sequence of any one of Tables 2 to 7, and whose ability to inhibit the expression of the AGT gene do not differ by more than that of a self-contained complete Inhibition of about 5, 10, 15, 20, 25 or 30% of the dsRNA of the sequence is considered to be within the scope of the invention.

In addition, the RNAs provided in Tables 2 to 7 identify sites in the AGT transcript that are susceptible to RISC-mediated cleavage. Accordingly, the invention further features an iRNA targeting one of these sites. As used herein, an iRNA is said to target a specific site on an RNA transcript if it promotes cleavage of the transcript anywhere within the specific site. Such iRNA will generally include at least about 19 contiguous nucleotides in the sequence provided in any one of Tables 2 to 7, with additional nucleotides taken from the region adjacent to the selected sequence in the AGT gene Acid sequence coupling.

III. Modified iRNA of the present disclosure

In some embodiments, the RNA of the iRNA of the invention is unmodified, e.g., dsRNA, and does not contain, e.g., chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of the iRNA of the invention is chemically modified to enhance stability or other beneficial properties, e.g., dsRNA. In some embodiments of the invention, substantially all nucleotides of the iRNA of the present disclosure are modified. In other embodiments of the invention, all nucleotides of the iRNA or substantially all nucleotides of the iRNA are modified, that is, no unmodified nucleotides are present in more than 5,000 strands of the iRNA. 4, 3, 2 or 1.

The nucleic acids featured in the present invention can be synthesized or modified by methods well established in the art, such as in "Current protocols in nucleus chemistry", Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, Those described in NY, USA are incorporated herein by reference. Modifications include, for example, terminal modifications, for example, 5'-end modifications (phosphorylation, conjugation, reverse linkage) or 3'-end modifications (conjugation, DNA nucleotides, reverse linkage, etc.); base modifications , e.g., substitution with stabilizing bases, destabilizing bases, or base pairing with an expanded resource library, removal of bases (abasic nucleotides) or conjugate bases; sugar modifications (e.g., in 2 '-position or 4'-position) or sugar substitution; or backbone modification, including modification or substitution of phosphodiester links. Specific examples of iRNA compounds useful in embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking native internucleoside linkages. RNAs containing modified backbones include especially those without phosphorus atoms in the backbone. For the purposes of this specification, and as is sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone may also be considered to be oligonucleosides. In some embodiments, the modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkyl phosphonates, including 3'-Alkylene phosphonates and chiral phosphonates, phosphonites, and amino phosphates include 3'-amino amino phosphates, amino alkyl amino phosphates, and thioamido phosphates Esters, thioalkyl phosphonates, thioalkyl phosphate triesters and boronic acid phosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and the adjacent nucleosides therein Unit pairs are connected 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts and free acid forms. In some embodiments of the invention, the dsRNA agents of the invention are in the free acid form. In other embodiments of the invention, the dsRNA agents of the invention are in salt form. In one embodiment, the dsRNA reagent of the invention is in the form of sodium salt. In certain embodiments, when the dsRNA agent of the invention is in the form of a sodium salt, sodium ions are present in the agent as counterions, and substantially all of the phosphodiester and/or phosphorothioate groups are present in the agent. . Agents in which substantially all phosphodiester and/or phosphorothioate linkages have sodium counterions, including no more than 5, 4, 3, 2, or 1 phosphodiester and/or sulfide linkages without sodium counterions. Phosphate linkage. In some embodiments, when the dsRNA agent of the present disclosure is in the sodium salt form, the sodium ions are present in the agent as counterions to all phosphodiester and/or phosphorothioate groups present in the agent .

Representative U.S. patents teaching preparation of the phosphorus-containing linkages described above include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,3 21,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,1 60,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,8 78,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464, the entire contents of which are hereby incorporated by reference. .

Modified RNA backbones that do not contain phosphorus atoms have structures composed of short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heterogeneous A skeleton formed by links between atoms or heterocyclic nucleosides. These include those containing morpholino linkages (formed in part from the sugar moieties of nucleosides); siloxane backbones; sulfide, trisine and trisulfide backbones; forming acetyl and thioacetyl backbones; methylene methane acyl and thiomethylacetyl skeletons; olefin-containing skeletons; amine sulfonate skeletons; methylene imino and methylene hydrazine skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and others containing mixed N , O, S and CH ₂ components.

Representative U.S. patents teaching preparation of the above-described oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,46 6,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; .

Suitable RNA mimetics are contemplated for use with the iRNAs provided herein, in which both the sugars of the nucleotide units and the internucleoside links, ie, the backbone, are replaced with new groups. Base units are maintained for hybridization to appropriate nucleic acid target compounds. One such oligomeric compound, in which RNA mimics have been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide containing backbone, specifically the amine ethyl glycine backbone phase. The nucleobase is retained and bound directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated by reference. Additional PNA compounds suitable for use in iRNAs of the present invention are described, for example, in Nielsen et al. , Science , 1991, 254, 1497-1500.

Some characteristic embodiments of the present invention include RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones, particularly -CH2 _- NH-- _CH2- , _-CH2 -N (CH ₃ )--O--CH ₂ --[called methylene (methyl imino group) or MMI skeleton], --CH ₂ --O--N(CH ₃ )--CH ₂ - The above-mentioned U.S. Patent No. 5,489,677 of -, --CH ₂ --N(CH ₃ )--N(CH ₃ )--CH ₂ -, and --N(CH ₃ )--CH ₂ --CH ₂ --, And the amide skeleton of the above-mentioned US Pat. No. 5,602,240. In some embodiments, the RNAs herein have the N-morpholine backbone structure of the above-mentioned US Pat. No. 5,034,506. The natural phosphodiester skeleton can be expressed as OP(O)(OH)-OCH2-

Modified RNA may also contain one or more substituted sugar moieties. The iRNAs featured herein, such as dsRNA, may include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S - or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and Alkynyl. Exemplary suitable modifications include O[(CH ₂ )nO]mCH ₃ , O(CH ₂ ).nOCH ₃ , O(CH ₂ )nNH ₂ , O(CH ₂ ) _n CH ₃ , O(CH ₂ )nONH ₂ and O(CH ₂ ) _n ON[(CH2)nCH ₃ )] ₂ , where n and m range from 1 to about 10. In other embodiments, the dsRNAs include one of the following at the 2' position: C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aryl Alkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkane Aryl group, aminoalkylamino group, polyalkylamino group, substituted silyl group, RNA cleavage group, reporter group, intercalator, group that improves iRNA pharmacokinetic properties or improves iRNA pharmacodynamics groups with similar properties, and other substituents with similar properties. In some embodiments, modifications include 2'-O--CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2 '-MOE) (Martin et al. , Helv. Chim. Acta , 1995, 78: 486-504), that is, alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., the O( _CH2 ) _2ON ( _CH3 ) ₂ group, also known as 2'-DMAOE, as shown in the examples below. as described above, and 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O- -CH ₂ --O--CH ₂ --N(CH ₃ ) ₂ . Other exemplary modifications include: 5'-Me-2'-F nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxynucleotide (these three families R and S isomers in ); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

Other modifications include 2'-methoxy (2'-OCH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions of the iRNA RNA, especially the 3' position of the sugar on the 3' terminal nucleotide or the 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide. The iRNA can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranose. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576 ,427;5,591,722;5,597,909;5,610,300;5,627,053;5,639,873 ; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are co-owned with instant applications. The entire contents of the foregoing are hereby incorporated by reference.

iRNA may also contain nucleobase (often referred to as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, Xanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy other 8-substituted adenine and guanine, 5- Halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8- Azaadenine, 7-desazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al. , Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, Y. Those disclosed in S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in this disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0 to 6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propyluracil Alkynylcytosine. 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993 , pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of the above-described modified nucleobases and other modified nucleobase moieties include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,1 66,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each This article is incorporated herein by reference.

In some embodiments, the RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. "Bicyclic sugar" is a modified furanosyl ring formed by a bridge between two carbons (whether adjacent or non-adjacent). A "bicyclic nucleoside"("BNA") is a nucleoside with a sugar moiety consisting of two carbons (whether adjacent or non-adjacent) that bridge the sugar ring. Dual loop system. In some embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring, optionally via the 2'-acyclic oxygen atom. Accordingly, in some embodiments, agents of the present disclosure may include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. In other words, LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'- _CH2 -O-2' bridge. This structure effectively "locks" ribose in the 3'-terminal structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al. , (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al. . , (2007) Mol Canc Ther 6(3): 833-843; Grunweller, A. et al. , (2003) Nucleic Acids Research 31(12): 3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present disclosure include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, antisense polynucleotide reagents of the present disclosure include one or more bicyclic nucleosides containing a 4' to 2' bridge.

Locked nucleosides can be represented by structures (stereochemistry omitted),

Among them, B is a nucleobase or a modified nucleobase, and L is a linking group connecting the 2'-carbon to the 4'-carbon of the ribose ring. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2';4' -(CH ₂ ) ₂ -O-2'(ENA);4'-CH(CH ₃ )-O-2' (also called "constrained ethyl" or "cEt") and 4 '-CH(CH ₂ OCH ₃ )-O-2' (and its analogs; see U.S. Patent No. 7,399,845); 4'-C(CH ₃ )(CH ₃ )-O-2' (and its analogs ; see, e.g., U.S. Patent No. 8,278,283); 4'-CH ₂ -N(OCH ₃ )-2' (and analogs thereof; see U.S. Patent No. 8,278,425); 4'-CH ₂ -ON(CH ₃ )-2' (See, e.g., U.S. Patent Publication No. 2004/0171570); 4'- _CH2 -N(R)-O-2', wherein R is H, C1-C12 alkyl, or nitrogen protecting group (see, e.g., U.S. Patent No. 7,427,672); 4'- _CH2 -C(H)( _CH3 )-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem. , 2009, 74, 118-134); and 4' _-CH2 -C(= _CH2 )-2' (and the like; see, eg, US Patent No. 8,278,426). The entire contents of the foregoing are hereby incorporated by reference.

Additional representative US patents and US patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: US Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 45;7,427,672;7,569,686 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; U.S. Patent Publication Nos. 2008/0039618; and 2009/0012281, the entire contents of each of which are hereby incorporated by reference.

Any of the above bicyclic nucleosides can be prepared containing one or more stereochemical sugar configurations including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to contain one or more restricted ethyl nucleotides. As used herein, "constrained ethyl nucleotide" or "cEt" includes nucleotides containing a 4'-CH(CH ₃ )-O-2' bridge (i.e., the L in the preceding structure). Locked nucleic acid with bicyclic sugar moiety. In one embodiment, the constrained ethyl nucleotide is in the S conformation, referred to herein as "S-cEt."

The iRNA of the invention may also contain one or more "conformationally restricted nucleotides" ("CRN"). CRNs are nucleotide analogs having linkers connecting the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRN locks the ribose ring into a stable conformation, increasing hybridization affinity with mRNA. The linkers are long enough to place oxygen in an optimal position for stability and affinity, thereby reducing ribose ring puckering.

Representative disclosures teaching preparation of portions of the CRN described above include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT Publication WO 2013/036868, the entire contents of each of which are hereby incorporated by reference.

In some embodiments, iRNAs of the invention comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any sugar bonds have been removed, forming an unlocked "sugar" residue. In one embodiment, UNA also includes monomers in which the bond between C1' to C4' has been removed (ie, the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another embodiment, the C2' to C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008 ) and Fluiter et al. , Mol. Biosyst., 2009, 10, 1039 are hereby incorporated by reference).

Representative U.S. publications teaching methods of preparing UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of which are incorporated herein by reference.

Possible stabilizing modifications to the ends of RNA molecules include N-(acetylhexyl)-4-hydroxyprolinol (Hy-C6-NHAc), N-(hexyl-4-hydroxyproline) ( Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline) (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoic acid) (Hyp-C6-amino), 2-docanoyl-uridine-3'-phosphate, reverse 2'-deoxy modified ribonucleotide, Examples include reverse dT (idT), reverse dA (idA), reverse abasic 2'-deoxyribonucleotide (iAb), etc. Disclosure of such modifications can be found in WO 2011/005861 .

In one embodiment, the 3' or 5' end of the oligonucleotide is connected to a reverse 2'-deoxy modified ribonucleotide, such as reverse dT (idT), reverse dA (idA ) or the reverse None Base 2’-deoxyribonucleotide (antibody). In a specific embodiment, a reverse 2'-deoxy modified ribonucleotide is attached to the 3' end of the oligonucleotide, such as the 3' end of the sense strand as described herein, wherein the attachment is via 3'-3'phosphodiester linkage or 3'-3'-phosphorothioate linkage.

In another embodiment, the 3'-terminus of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3'-phosphorothioate linkage. In another embodiment, the 3'-terminus of the sense strand is linked to reverse dA (idA) via a 3'-3'-phosphorothioate linkage.

In a specific embodiment, a reverse 2'-deoxy modified ribonucleotide is attached to the 3' end of the oligonucleotide, such as the 3' end of the sense strand as described herein, wherein the attachment is via 3'-3'phosphodiester linkage or 3'-3'-phosphorothioate linkage.

In another embodiment, the 3'-terminal nucleotide of the sense strand is inverted dA (idA) and is linked via a 3'-3'-link (e.g., 3'-3'-phosphorothioate ester link) to the preceding nucleotide.

Other modifications to the nucleotides of the iRNAs of the present disclosure include 5' phosphates or 5' phosphate mimetics, e.g., 5'-terminal phosphates or phosphate mimetics on the antisense strand of the iRNA. Suitable phosphate mimetics are disclosed, for example, in US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNA containing the motif of the invention

In certain aspects of the invention, the double-stranded RNA agents of the invention include agents with chemical modifications as disclosed in, for example, PCT Application No. PCT/ titled "Modified Double Stranded Oligonucleotides" filed on October 28, 2021. US2021/057016 (Attorney Docket No. ALN-384WO), the entire contents of which are incorporated herein by reference.

In some aspects of the invention, double-stranded RNA agents of the present disclosure include agents with chemical modifications as disclosed, for example, in WO2013/075035, the entire contents of each of which are incorporated by reference. Enter this article. As shown herein and in WO2013/075035, one or more motifs of three identical modifications on three consecutive nucleotides can be introduced into the sense or antisense strand of a dsRNAi agent, specifically tethered at the cleavage site or nearby. In some embodiments, the sense and antisense strands of the dsRNAi agent can additionally be completely modified. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present. The dsRNAi agent can optionally be coupled to a GalNAc-derived ligand, for example on the sense strand.

More specifically, when the sense and antisense strands of the double-stranded RNA agent are fully modified to have three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent Gene-silencing activity of dsRNAi agents is observed when targeting one or more motifs.

Therefore, the present invention provides a double-stranded RNA agent capable of inhibiting the expression of a target gene (ie, AGT gene) in vivo. The RNAi agent contains a sense strand and an antisense strand. Each strand of the RNAi agent can be, for example, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length or 21 to 23 nucleotides in length.

The sense and antisense strands typically form a duplex double-stranded RNA ("dsRNA"), also referred to herein as a "dsRNAi agent." The double-stranded region of the dsRNAi agent can be, for example, the double-stranded region can be 27 to 30 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to a length of 21 nucleotide pairs, a length of 21 to 25 nucleotide pairs, or a length of 21 to 23 nucleotide pairs. In another embodiment, the duplex region is selected from the group consisting of 19, 20, 21, 22, 23, 24, 25, 26 and 27 nucleotides in length.

In some embodiments, the dsRNAi agent may include one or more overhangs, either at the 3'-end or the 5'-end, or at both ends of one or both strands. The overhang can independently be from 1 to 6 nucleotides in length, for example from 2 to 6 nucleotides in length, from 1 to 5 nucleotides in length, from 2 to 5 nucleotides in length, from 1 to 4 nucleotides in length. Nucleotide length, 2 to 4 nucleotides in length, to 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, 1 to 2 nucleotides in length. In some embodiments, the protruding area may include an expanded protruding area as provided above. This protrusion can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a genetic mismatch with the target mRNA, or it can be complementary to the gene sequence being targeted, or it can be linked to another sequence. The first and second strands may also be connected, for example, by additional bases forming hairpins, or by other non-base linkers.

In certain embodiments, the nucleotides in the protruding region of the dsRNAi agent can each independently be a modified or unmodified nucleotide, including, but not limited to, 2'-sugar modified, e.g., 2' -F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2`-O-methoxyethyl-5-methylcytidine (m5Ceo) and any combination thereof.

For example, TT can be an overhang sequence on either end of either strand. The overhang can form a genetic mismatch with the target mRNA, or it can be complementary to the gene sequence being targeted, or it can be linked to another sequence.

The 5′- or 3′-overhangs on the sense strand, antisense strand, or both strands of the dsRNAi agent may be phosphorylated. In some embodiments, the overhang region includes two nucleotides with a phosphorothioate between them, wherein the two nucleotides may be the same or different. In some embodiments, the protrusion is present at the 3′-end of the sense strand, the antisense strand, or both strands. In some implementations, the 3'-projection is present in the antisense strand. In some implementations, the 3'-protrusion is present in the sense stock.

The dsRNAi agent may contain only a protrusion that enhances the interfering activity of the RNAi without affecting its overall stability. For example, the single-strand protrusion may be located at the 3'-end of the sense strand or, alternatively, at the 3'-end of the antisense strand. The RNAi may also have a blunt end located at the 5'-end of the antisense strand (i.e., the 3'-end of the sense strand), or vice versa. Typically, the antisense strand of a dsRNAi agent has a nucleotide overhang at the 3’-end and a blunt end at the 5’-end. While not wishing to be bound by theory, the asymmetric blunt end of the 5'-end of the antisense strand and the overhang of the 3' end of the antisense strand facilitate loading of the leader strand into the RISC process.

In some embodiments, the dsRNAi agent is double blunt-ended 19 nucleotides in length, wherein the sense strand is comprised of three consecutive nucleotides from positions 7, 8, and 9 from the 5' end. At least one motif consisting of three 2'-F modifications. The antisense strand contains at least one motif consisting of three 2'-O-methyl modifications at three consecutive nucleotides from the 5' end at positions 11, 12 and 13.

In other embodiments, the dsRNAi agent is bi-blunt-ended in length of 20 nucleotides, wherein the sense strand contains at least A motif consisting of three 2'-F modifications. The antisense strand contains at least one motif consisting of three 2'-O-methyl modifications at three consecutive nucleotides from the 5' end at positions 11, 12 and 13.

In some embodiments, the dsRNAi agent is a double-blunt end of 21 nucleotides in length, wherein the sense strand contains at least A motif consisting of three 2'-F modifications. The antisense strand contains at least one motif consisting of three 2'-O-methyl modifications at three consecutive nucleotides from the 5' end at positions 11, 12 and 13.

In some embodiments, the dsRNAi agent includes a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand is included at positions 9, 10, and 11 from the 5' end. The three consecutive nucleotides of the antisense strand contain at least one motif consisting of three 2'-F modifications; the antisense strand contains at least one of the three consecutive nucleotides at positions 11, 12 and 13 from the 5' end A motif consisting of three 2'-O-methyl modifications, while the other end contains a 2-nucleotide overhang. In one embodiment, the 2-nucleotide overhang is located at the 3'-end of the antisense strand.

When 2 nucleotides protrude from the 3' end of the antisense strand, there may be two phosphorothioate internucleotide links between the three nucleotides at the end, where two of the three nucleotides Each is the overhanging nucleotide, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent has an additional two phosphorothioate internucleotide linkages between the three nucleotides at the 5'-end of the sense strand and the 5'-end of the antisense strand. . In some embodiments, each nucleotide in the sense and antisense strands of the dsRNAi agent, including nucleotides that are part of the motif, is a modified nucleotide. In some embodiments, each residue is independently modified with 2'-O-methyl or 3'-fluoro, e.g., in an alternating motif, and optionally, the dsRNAi agent further includes a ligand (e.g., GalNAc ₃ ).

In some embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length, starting from the 5' terminal nucleotide (position 1) , positions 1 to 23 of the first strand contain at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, starting from the 3' terminal nucleotide, at the same position as the sense strand Paired positions 1 to 23 contain at least 8 ribonucleotides, forming a duplex; in which at least the 3' terminal nucleotides of the antisense strand are unpaired with the sense strand, and up to 6 consecutive 3' The terminal nucleotide is unpaired with the sense strand, resulting in a 3' single-stranded overhang of 1 to 6 nucleotides; the 5' of the antisense strand The terminal contains 10 to 30 consecutive nucleotides that do not pair with the sense strand, resulting in a single-stranded 5' overhang of 10 to 30 nucleotides; where the sense and antisense strands are aligned for maximum complementarity When at least the 5' terminal and 3' terminal nucleotides of the sense strand are base-paired with the nucleotides of the antisense strand, a substantially double-stranded region is formed between the sense strand and the antisense strand; when the double-stranded When the nucleic acid is introduced into mammalian cells, the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides in length of the antisense strand to reduce target gene expression; and the sense strand is located within three consecutive nucleotides Contains at least one motif consisting of three 2'-F modifications, at least one of which occurs at or near the cleavage site. The antisense strand contains at least one motif consisting of three 2'-O-methyl modifications in three consecutive nucleotides at or near the cleavage site.

In some embodiments, the dsRNAi agent includes a sense strand and an antisense strand, wherein the dsRNAi agent includes a first strand that is at least 25 and at most 29 nucleotides in length and a first strand that is at most 30 nucleotides in length and A second strand containing at least one motif consisting of three 2'-O-methyl modifications at positions 11, 12 and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand to form a blunt end, the 3' end of the second strand is 1 to 4 nucleotides longer than the first strand, wherein the duplex region is at least 25 nucleotides in length, when When an RNAi agent is introduced into a mammalian cell, the second strand is sufficiently complementary to at least 19 nucleotides of target mRNA along the length of the second strand and reduces target gene expression, and wherein Dicer cleavage of the dsRNAi agent produces a second strand containing siRNA at the 3'-end of the strand, thereby reducing the expression of the target gene in mammals. Optionally, the dsRNAi agent also contains a ligand.

In some embodiments, the sense strand of the dsRNAi agent contains at least one motif consisting of three identical modifications on three consecutive nucleotides, wherein one of the motifs occurs at the cleavage site of the sense strand.

In some embodiments, the antisense strand of the dsRNAi agent may further comprise at least one motif consisting of three identical modifications on three consecutive nucleotides, wherein one of the motifs appears at the cleavage site in the antisense strand. at or near the point.

For dsRNAi agents containing duplex regions of 19 to 23 nucleotides in length, the cleavage sites of the antisense strand are typically near positions 10, 11, and 12 from the 5'-end. Therefore, a motif with three identical modifications may appear at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14 of the antisense strand , position 15, calculated from the first nucleotide at the 5'-end of the antisense strand, or from the first paired nucleotide in the duplex region at the 5'-end of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the duplex region of the dsRNAi agent starting from the 5’-end.

The sense strand of the dsRNAi agent may contain at least one motif consisting of three identical modifications at three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may be at or near the cleavage site of the strand Three consecutive nucleotides have at least one motif consisting of three identical modifications. When the sense and antisense strands form a dsRNA duplex, the sense and antisense strands can be aligned, i.e. the sense strand consists of a motif of three nucleotides and the antisense strand consists of three nucleosides A motif composed of acids has at least one base overlap, that is, at least one of the three bases of the sense strand motif forms a base pair with at least one of the three bases of the antisense strand motif. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may contain more than one motif consisting of three identical modifications on three consecutive nucleotides. The first motif can occur at or near the cleavage site of the strand, while other motifs can be winged. The term "wing modification" as used herein refers to the occurrence of separation from the motif at or near the cleavage site of the same strand the other part of the strand. The wing modification is adjacent to the first motif or separated by at least one or more nucleotides. The chemistry of the motifs differs from each other when they are directly adjacent to each other, and the chemistry can be the same or different when the motifs are separated by one or more nucleotides. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification can occur at the terminus relative to the first motif at or near the cleavage site, or on either side of the leading motif.

As with the sense strand, the antisense strand of the dsRNAi agent may contain more than one motif consisting of three identical modifications on three consecutive nucleotides, at least one of which occurs at or near the cleavage site of the strand . The antisense strand may also contain one or more wing modifications arranged in a manner similar to the wing modifications that may be present on the sense strand.

In some embodiments, wing modifications of the sense or antisense strand of the dsRNAi agent generally do not include the first or both nucleotides at the 3'-end, 5'-end, or both ends of the strand.

In other embodiments, the wing modifications of the sense strand or antisense strand of the dsRNAi agent generally do not include the first or both ends of the strand at the 3'-end, 5'-end, or both ends of the duplex region Paired nucleotides.

When the sense and antisense strands of the dsRNAi agent each comprise at least one wing modification, the wing modifications can fall on the same end of the duplex region and overlap by one, two, or three nucleotides.

When the sense strand and antisense strand of the dsRNAi agent each contain at least two wing modifications, the sense strand and antisense strand can be arranged such that the two modifications from one strand each fall on one end of the duplex region , containing an overlap of one, two or three nucleotides; with two modifications from one strand falling on the other end of the duplex region, with an overlap of one, two or three nucleotides; one strand two Modifications fall on each side of the leader motif, with one, two, or three nucleotide overlap in the duplex region.

In some embodiments, each nucleotide in the sense and antisense strands of the dsRNAi agent, including nucleotides that are part of the motif, can be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more changes to one or more non-linked phosphate oxygens or one or more linked phosphate oxygens; altering the composition of the ribose, e.g. 2' -hydroxyl group; total substitution of phosphate with a "dephospho"linker; modification or substitution of naturally occurring bases; and substitution or modification of the ribose-phosphate backbone.

Since nucleic acids are polymers of subunits, many modifications occur at repeated positions within the nucleic acid, for example, base modifications, or phosphate moieties, or non-linked O's of the phosphate moiety. In some cases, modifications will occur at all subject positions in the nucleic acid, but in many cases not. By way of example, modifications may occur only at the 3'- or 5'-terminal position, may occur only at the terminal region, for example, at the terminal nucleotide position or the last 2, 3, 4, 5 or 10 of a strand. Nucleotides. Modifications may occur in the double-stranded region, the single-stranded region, or both. Modifications may occur only in double-stranded regions of RNA or may occur only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions may occur only at one or both termini, and may occur only in the terminal region, e.g., at the terminal nucleotide position or at the last 2, 3, 4, 5 of a strand or in 10 nucleotides, or may occur in double-stranded and single-stranded regions, especially at the ends. The 5'-end or terminus can be phosphorylated.

It may be possible, for example, to increase stability by including specific bases in the overhangs, or by including modified nucleotides or nucleotide substitutions in the single-stranded overhangs, for example, in the 5'- or 3'-overhangs, or in the Of the two. For example, it may be necessary to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3'- or 5'-overhang can be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications known in the art at the 2' position of ribose, for example, the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2' -O-methyl modification of ribose without a nucleobase, and modification of the phosphate group, for example, phosphorothioate modification. The overhang need not be homologous to the target sequence.

In some implementations, each residue of the sense and antisense strands is independently modified by LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2 '-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxy or 2'-fluoro. Strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

There are usually at least two different modifications on the sense and antisense strands. These two modifications can be 2'-O-methyl or 2'-fluoro modification or others.

In some implementations, the Na or _{N b includes} modifications in alternating modes. As used herein, the term "alternating motif" refers to a motif containing one or more modifications, each modification occurring on a strand of alternating nucleotides. The alternating nucleotides may refer to every other nucleotide or every third nucleotide, or a similar pattern. For example, if A, B, and C each represent a modification of a nucleotide, the alternating motif could be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB..."..." or "ABCABCABCABC..." etc.

The types of modifications included in alternating motifs can be the same or different. For example, if A, B, C, and D each represent a modification of one nucleotide, the alternating pattern (i.e., modification of every other nucleotide) may be the same, but each may be chosen to be derived from several nucleotides within the alternating motif. modification possibilities Meaningful or inverse stocks, such as "ABABABAB...", "ACACAC...", "BDBDBD..." or "CDCCDD..." etc.

In some embodiments, the dsRNAi agents of the invention comprise a modification pattern of alternating motifs on the sense strand that is displaced relative to the modification pattern of alternating motifs on the antisense strand. The shift can cause a modified set of nucleotides on the sense strand to correspond to a different modified set of nucleotides on the antisense strand, and vice versa. For example, when a sense strand is paired with an antisense strand in a dsRNA duplex, the alternating motif in the sense strand could begin with "ABABAB" from 5' to 3' of the strand, while the alternating motif in the antisense strand The motif can start with "BABABA" from 5' to 3' of the strand within the double-stranded region. As another example, the alternating motifs in the sense strand can start with "AABBAABB" from 5' to 3' of the strand in the double-stranded region, and the cross-motif sequence in the antisense strand can start from 5' to 3' of the strand in the double-stranded region. 5' to 3' starts with "BBAABBAA". Double-stranded regions, and therefore the modification pattern between the sense and antisense strands, are completely or partially shifted.

In a specific embodiment, the alternating motif system in the sense strand is a self-stranded 5'3' "ABABAB", where each A is an unmodified ribonucleotide and each B is a 2' -O-methyl modified nucleotides.

In a specific embodiment, the alternating motif system in the sense strand is a self-stranded 5'3' "ABABAB", wherein each A is a 2'-deoxy-2'-fluoro modified nucleotide, And each B is a 2'-O methyl modified nucleotide.

In another specific embodiment, the alternating motif system in the antisense strand is composed of self-stranded 3' to 5' "BABABA", wherein each A is a 2'-deoxy-2'-fluoro modified nucleoside Acid, each B is a 2'-O methyl modified nucleotide.

In a specific embodiment, the alternating pattern system in the sense strand is "ABABAB" starting at strand 5'3', while the alternating pattern system in the antisense strand is starting at strand 3'-5' "BABAB", wherein each A is an unmodified ribonucleotide and each B is a 2'-Omethyl modified nucleotide.

In a specific embodiment, the alternating pattern system in the sense strand is "ABABAB" starting at strand 5'3', while the alternating pattern system in the antisense strand is starting at strand 3'-5' with "BABAB" ", where each A is a 2'-deoxy-2'-fluoro modified nucleotide, and each B is a 2'-Omethyl modified nucleotide.

In some embodiments, the dsRNAi agent comprises a pattern of alternating motifs of 2'-O-methyl modifications and 2'-F modifications on the sense strand, initially relative to the pattern of alternating motifs of 2'-O- Has an offset. Initially, methyl modification and 2'-F modification are performed on the antisense strand, i.e., 2'-O-methyl modified nucleotides on the sense strand and 2'-F modified nucleotide bases on the antisense strand Yes, vice versa. Position 1 of the sense strand can start with a 2'-F modification, and position 1 of the antisense strand can start with a 2'-O-methyl modification.

The introduction of one or more motifs consisting of three identical modifications on three consecutive nucleotides into the sense or antisense strand disrupts the initial modification pattern present in the sense or antisense strand. This modification pattern, which interrupts the sense or antisense strand by introducing one or more motifs consisting of three identical modifications into three consecutive nucleotides into the sense or antisense strand, can enhance the targeting of target genes. gene silencing activity.

In some embodiments, when a motif consisting of three identical modifications for three consecutive nucleotides is introduced into any strand, the modification of the nucleotide immediately adjacent to the motif is different from the modification of the motif. For example, the portion of the sequence that contains the motif is "...N _a YYYN _b ...", where "Y" represents the modification of the motif on three consecutive nucleotides with three identical modifications, "N _a " and "N _b ” _{represents the modification of the nucleotide next to the motif “YYY”, which “YYY” is different from the modification of Y, and Na and N b can} be the same or different modifications. Alternatively, when wing modifications are present, _Na or _Nb may be present or absent.

The iRNA may comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide link modification can occur on any nucleotide at any position on the sense strand, the antisense strand, or both strands. For example, the internucleotide link modification may occur on each nucleotide of the sense strand or the antisense strand; each internucleotide link modification may occur on the sense strand or the antisense strand in an alternating pattern; Alternatively, the sense or antisense strand may contain two internucleotide linkage modifications in an alternating pattern. The alternating pattern of inter-nucleotide link modifications in the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of inter-nucleotide link modifications in the sense strand may be different than that of the nucleotides in the antisense strand. The offset of the alternating pattern of inter-link modifications. In one embodiment, the double-stranded RNAi agent contains 6 to 8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand contains two phosphorothioate internucleotide linkages at the 5'-end and two phosphorothioate internucleotide linkages at the 3'-end, and has The sense strand contains at least two phosphorothioate internucleotide links at the 5'-terminus or the 3'-terminus.

In some embodiments, the dsRNAi agent contains a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may comprise two nucleotides with a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications can also be performed to connect overhanging nucleotides to end-paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all of the overhanging nucleotides may be linked by phosphorothioate or methylphosphonate internucleoside links, and if desired, additional phosphorothioate or methyl groups may be present. Phosphonate internucleotide links connect overhanging nucleotides to adjacent paired nucleotides. For example, there may be at least two phosphorothioate internucleotide links between the three nucleotides at the Two of them are overhanging nucleotides, and the third is the paired nucleotide immediately adjacent to the overhanging nucleotide. The three nucleotides at these ends may be located at the 3'-end of the antisense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, or the 5'-end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is located at the 3'-end of the antisense strand, with two phosphorothioate internucleotide links between the three nucleotides at the end, three of which are Two of the nucleotides are the overhanging nucleotides, and the third nucleotide is the paired nucleotide next to the overhanging nucleotide. Optionally, the dsRNAi agent may additionally have two phosphorothioate internucleotide links between the three nucleotides at the 5'-end of the sense strand and the 5'-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises a mismatch to the target (within a duplex) or a combination thereof. This mismatch can occur in the overhang region or in the duplex region. The base pairs can be ordered according to their propensity to promote dissociation or melting (e.g., according to the binding or dissociation free energy of a particular pair). The simplest approach is to examine these pairs on a single pairing basis, although the next adjacent or similar analysis can also be used). In promoting dissociation: A:U is better than G:C; G:U is better than G:C; and I:C is better than G:C (I=inosine). Mismatches, for example, non-canonical or non-canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairs containing universal bases are preferred over canonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region starting from the 5'-end of the antisense strand, independently selected from : Groups of A:U, G:U, I:C and mismatched pairs, for example, non-canonical or non-canonical pairings or pairs containing universal bases to facilitate antisense strands at the 5' end of the duplex dissociation.

In some embodiments, the nucleotide at position 1 within the duplex region starting from the 5'-end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, from the 5’ end of the antisense strand At least one of the first 1, 2 or 3 base pairs within the initial duplex region is an AU base pair. For example, the first base pair within the duplex region starting from the 5' end of the antisense strand is an AU base pair.

In other embodiments, the 3'-terminal nucleotide of the sense strand is deoxythymidine (dT) or the 3'-terminal nucleotide of the antisense strand is deoxythymidine (dT). For example, there is a short sequence of deoxythymidine nucleotides, e.g., two dT nucleotides at the 3'-end of the sense strand, the antisense strand, or both strands.

In some implementations, the beneficial stock sequence can be represented by formula (I):

5' n _p -N _a -(XXX) _i -N _b -YYY-N _b -(ZZZ) _j -N _a -n _q 3' (I)

in:

i and j are each independently 0 or 1;

p and q each independently range from 0 to 6;

_Na each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and the sequences each contain at least two differently modified nucleotides;

Nb each independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides;

n _p and n _q independently represent overhanging nucleotides;

where Nb and Y do not have the same modification; and

XXX, YYY and ZZZ respectively represent three consecutive nucleotides consisting of three identical modifications. In an example embodiment, YYY are 2'-F modified nucleotides.

In some embodiments, the Na or _{N b include} alternating patterns of modifications.

In some embodiments, the YYY motif is present at or near the cleavage site of the sense strand. For example, when the dsRNAi agent has a duplex region ranging from 17 to 23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., can occur At positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13), counting from the first nucleotide, Starting from the 5'-end; or, if appropriate, counting from the first paired nucleotide within the duplex region, starting from the 5'-end.

In one implementation, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the equity shares can be expressed by the following formula:

5' n _p -N _a -YYY-N _b -ZZZ-N _a -n _q 3'(Ib);

5' n _p -N _a -XXX-N _b -YYY-N _a -n _q 3'(Ic); or

5' n _p -N _a -XXX-N _b -YYY-N _b -ZZZ-N _a -n _q 3' (Id).

When the sense strand is represented by formula (Ib), _Nb represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. _Na may each independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the sense strand is represented by formula (Ic), N _b means containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides oligonucleotide sequence. _Na may each independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the sense strand is represented by formula (Id), N _b each independently represents an oligonucleotide comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides sequence. In one embodiment, N _b is 0, 1, 2, 3, 4, 5 or 6. _Na may each independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

Each X, Y and Z may be the same or different from each other.

In other implementations, i is 0 and j is 0, and the right shares can be represented by the following formula:

5' n _p -N _a -YYY-N _a -n _q 3' (Ia).

When the sense strand is represented by formula (Ia), _Na may each independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

In one embodiment, the RNAi antisense sequence can be represented by formula (II):

5' n _q' -N _a '-(Z'Z'Z') _k -N _b '-Y'Y'Y'-N _b '-(X'X'X') _l -N' _a -n _p '3' (II)

in:

k and l are each independently 0 or 1;

p' and q' are independently from 0 to 6;

N _a ' each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides;

N _b ' each independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides;

n _p ' and n _q ' each independently represent overhanging nucleotides;

where Nb' and Y' do not have the same modification; and

X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif consisting of three identical modifications on three consecutive nucleotides.

In some embodiments, the _Na ' or _Nb ' includes an alternating pattern of modifications.

The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNAi agent has a duplex region ranging from 17 to 23 nucleotides in length, the Y'Y'Y' motif can appear at positions 9, 10, 11; 10, 11, 12 of the antisense strand ; 11, 12, 13; 12, 13, 14; or 13, 14, 15, starting from the 5'-end, counting from the first nucleotide; or, if necessary, starting from the 5' within the duplex region - Counting begins with the first paired nucleotide starting from the end. In one embodiment, the Y'Y'Y' pattern appears at positions 11, 12, and 13.

In some embodiments, the Y'Y'Y' module system is fully modified with 2'-OMe. Nucleotides.

In some embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

Antisense stocks can be represented by the following formula:

5' n _q' -N _a '-Z'Z'Z'-N _b '-Y'Y'Y'-N _a '-n _p '3'(IIb);

5' n _q' -N _a '-Y'Y'Y'-N _b '-X'X'X'-n _p '3'(IIc); or

5'n _q' -N _a '-Z'Z'Z'-N _b '-Y'Y'Y'-N _b '-X'X'X'-N _a '-n _p '3' (IId ).

When the antisense strand is represented by formula (IIb), N _b ' means comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleosides acid oligonucleotide sequence. _Na ' each independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the antisense strand is represented by formula (IIc), N _b ' means comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleosides acid oligonucleotide sequence. N _a ' each independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the antisense strand is represented by formula (IId), N _b ' each independently represents 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 For an oligonucleotide sequence of modified nucleotides, _Na ' each independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15 or 2 to 10 modified nucleotides. In one embodiment, N _b is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand can be represented by the following formula:

5' n _p '-N _a '-Y'Y'Y'-N _a '-n _q '3' (Ia).

When the antisense strand is of formula (IIa), N _a ' each independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

Each X', Y' and Z' can be the same as or different from each other.

Each nucleotide of the sense and antisense strands may be independently modified by LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O- Allyl, 2'-C-allyl, 2'-hydroxy or 2'-fluoro modification. For example, each nucleotide in the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y' and Z' may particularly represent a 2'-O-methyl modification or a 2'-fluoro modification.

In some embodiments, when the duplex region is 21 nt, the sense strand of the dsRNAi agent can comprise the YYY motif occurring at positions 9, 10, and 11 of the strand, starting from the 5'-end of the first core Counting begins with the nucleotide or, if appropriate, with the first paired nucleotide within the duplex region starting from the 5' end; Y indicates a 2'-F modification. The sense strand may additionally contain an XXX motif or a ZZZ motif as a wing modification at the other end of the double-stranded region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

In some embodiments, the antisense strand may comprise a Y'Y'Y' motif occurring at positions 11, 12, 13 counting from the first nucleotide at the 5'-end of the strand, or Required, counting starts from the first pair of nucleotides within the duplex region starting from the 5'-end; Y' represents a 2'-O-methyl modification. The antisense strand may additionally contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the other end of the duplex region; X'X'X' and Z'Z'Z' respectively independently represents 2'-OMe modification or 2'-F modification.

The shares represented by any of the above formulas (Ia), (Ib), (Ic) and (Id) are the same as those represented by any of the formulas (IIa), (IIb), (IIb), (IIc) and (IId). The antisense strands represented by the formula individually form a duplex.

Accordingly, a dsRNAi agent used in the methods of the present disclosure may comprise a sense strand and an antisense strand, each having 14 to 30 nucleotides, the iRNA duplex represented by formula (III):

Meaningful: 5' n _p -N _a -(XXX) _i -N _b -YY YN _b -(ZZZ) _j -N _a -n _q 3'

Antonym: 3' n _p ^' -N _a ^' -(X'X'X') _k -N _b ^' -Y'Y'Y'-N _b ^' -(Z'Z'Z') _l -N _a ^' -n _q ^' 5'(III)

in:

i, j, k, l are each independently 0 or 1;

p, p', q, q' are independently 0 to 6;

_Na and _Na ' each independently represent an oligonucleotide sequence containing 0 to 25 modified nucleotides, and the sequences each contain at least two different modified nucleotides;

Nb and Nb' each independently represent an oligonucleotide sequence containing 0 to 10 modified nucleotides;

wherein np', np, nq' and nq may each be present or absent and independently represent a protruding nucleotide; and

XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif consisting of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of sense and antisense strands to form iRNA duplexes include the following formula:

5' n _p -N _a -YY YN _a -n _q 3'

3' n _p ^' -N _a ^' -Y ' Y ' Y ' -N _a ^' n _q ^' 5'(IIIa)

5' n _p -N _a -YY YN _b -ZZ ZN _a -n _q 3'

3' n _p ^' -N _a ^' -Y ' Y ' Y ' -N _b ^' -Z ' Z ' Z ' -N _a ^' n _q ^' 5'(IIIb)

5' n _p -N _a -XX XN _b -YY YN _a -n _q 3'

3' n _p ^' -N _a ^' -X ' X ' X ' -N _b ^' -Y ' Y ' Y ' -N _a ^' -n _q ^' 5'(IIIc)

5' n _p -N _a -XX XN _b -YY YN _b -ZZ ZN _a -n _q 3'

3' n _p ^' -N _a ^' -X ' X ' X ' -N _b ^' -Y ' Y ' Y ' -N _b ^' -Z ' Z ' Z ' -N _a -n _q ^' 5'(IIId)

When the dsRNAi agent is represented by formula (IIIa), _Na each independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), Nb each independently represents an oligonucleotide sequence comprising 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Na _each independently represents an oligonucleotide sequence including 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIc), N _b and N _b ' each independently represent 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or an oligonucleotide sequence of 0 modified nucleotides. N _a each independently represents an oligonucleotide sequence including 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIId), N _b and N _b ' each independently represent 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or an oligonucleotide sequence of 0 modified nucleotides. _Na , _Na ' each independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15 or 2 to 10 modified nucleotides. Na, Na', Nb and Nb' each independently contain alternating patterns of modifications.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) may be the same as or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one Y nucleotide can form a base pair with one Y' nucleotide. Alternatively, at least two Y nucleotides form base pairs with corresponding Y' nucleotides; or all three Y nucleotides form base pairs with corresponding Y' nucleotides.

When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one Z nucleotide can form a base pair with one Z' nucleotide. Alternatively, at least two Z nucleotides form base pairs with corresponding Z' nucleotides; or all three Z nucleotides form base pairs with corresponding Z' nucleotides.

When the dsRNAi agent is represented by formula (IIIc) or (IIId), at least one X nucleotide can form a base pair with one X' nucleotide. Alternatively, at least two X nucleotides form base pairs with corresponding X' nucleotides; or all three X nucleotides form base pairs with corresponding X' nucleotides.

In some embodiments, when the modification of the Y nucleotide is different from the modification of the Y' nucleotide, the modification of the Z nucleotide is different from the modification of the Z' nucleotide, or the modification of the X nucleotide is different from the modification of the 'Nucleotides are modified differently.

In some embodiments, when the dsRNAi agent is represented by formula (IIId), the _Na modification is a 2' - O-methyl or 2' -fluoro modification. In other embodiments, when the RNAi agent is represented by Formula (IIId), the _Na modification is 2' - O-methyl or 2' -fluoro modification and n _p '>0 and at least one n _p ' is consistent with The adjacent nucleotides a are linked by phosphorothioates. In yet another embodiment example, when the RNAi agent is represented by formula (IIId), the _Na modification is 2' - O-methyl or 2' -fluoro modification, n _p '>0 and at least one n _p ' Linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand is conjugated to one or more GalNAc derivatives (described below) linked via divalent or trivalent branched linkers. In other embodiments, when the RNAi agent is represented by formula (IIId), the _Na modification is a 2' - O-methyl or 2' -fluoro modification, n _p '>0 and at least one n _p ' is Adjacent nucleotides are linked by phosphorothioate, and the sense strand contains at least one phosphorothioate link, and the sense strand is connected to one or more phosphorothioate linkers through divalent or trivalent branched linkers. GalNAc derivative conjugation.

In some embodiments, when the dsRNAi agent is represented by Formula (IIIa), the _Na modification is a 2' -O-methyl or 2' -fluoro modification, n _p '>0 and at least one n _p ' is Adjacent nucleotides are linked by a phosphorothioate link, the sense strand contains at least one phosphorothioate link, and the sense strand has one or more GalNAc derivatives attached by a divalent or trivalent branched linker Engagement.

In some embodiments, the dsRNAi agent is a polymer containing at least two duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes The bodies are connected by links. The linker may be cleavable or non-cleavable. Optionally, the multimer also contains a ligand. Each duplex can target the same gene or two different genes; or each duplex can target the same gene at two different target sites.

In some embodiments, the dsRNAi agent contains three, four, five, six or more of formulas (III), (IIIa), (IIIb), (IIIc) and (IIId). A multimer of duplexes in which the duplex systems are connected by linkers. The linker may be cleavable or non-cleavable. Optionally, the multimer also contains a ligand. Each duplex can target the same gene or two different genes; or each duplex can target the same gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) are connected to each other at the 5' end, and one or both 3' end and optionally conjugated to a ligand. Each agent can target the same gene or two different genes; or each agent can target the same gene at two different target sites.

In some embodiments, the RNAi agents of the invention may include a small number of nucleotides containing 2'-fluoro modifications, for example, 10 or less nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides bearing a 2'-fluoro modification. In a specific embodiment, the RNAi agent of the present disclosure contains 10 nucleotides with 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications in the sense strand and 4 nucleotides in the antisense strand. 6 nucleotide strands with 2'-fluoro modification. In another embodiment, the RNAi agent of the present disclosure contains 6 nucleotides with 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications in the sense strand and the antisense strand. 2 nucleotides with 2'-fluoro modifications.

In other embodiments, the RNAi agents of the invention may contain a very small number of nucleotides containing 2'-fluoro modifications, for example, 2 or less nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 2, 1, or 0 nucleotides with a 2'-fluoro modification. In one embodiment, the RNAi agent may contain 2 nucleotides with a 2'-fluoro modification, e.g., 0 nucleotides with a 2'-fluoro modification in the sense strand and 0 nucleotides in the antisense strand. 2 nucleotides with 2'-fluoro modification.

Various publications describe multimeric iRNAs useful in the methods of the invention. Such disclosures include WO2007/091269, US Patent No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520, the entire contents of each of which is hereby incorporated by reference.

In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In an exemplary embodiment, the 5'vinylphosphonate-modified nucleotide of the present disclosure has the following structure:

Where X is O or S;

R is hydrogen, hydroxyl, fluorine or C _1-20 alkoxy (such as methoxy or n-hexadecyloxy);

R5' is =C(H)-P(O)(OH) ₂ and the double bond between the C5' carbon and ^R5 ' is in the E or Z direction (e.g., E direction); and

B is a nucleobase or modified nucleobase, optionally, wherein B is adenine, guanine, cytosine, thymine or uracil.

In one embodiment, R ⁵ ' is =C(H)-P(O)(OH) ₂ , and the double bond between the C5' carbon and R5' is in the E direction. In another embodiment, R is a methoxy group, R ⁵ ' is =C(H)-P(O)(OH) ₂ , and the double bond between the C5' carbon and R5' is in the E direction. In another embodiment _, .

The vinyl phosphonate of the disclosure can be linked to the antisense or sense strand of the dsRNA of the disclosure. In some embodiments, the vinyl phosphonate of the present disclosure is linked to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

Vinyl phosphonate modifications are also contemplated for use in the compositions and methods of the present disclosure. Exemplary vinyl phosphonate structures include the aforementioned structures, wherein R5' is =C(H)-OP(O)(OH)2 and the double bond between the C5' carbon and R5' is in the E or Z direction (e.g., E direction).

As described in more detail below, iRNAs containing one or more carbohydrate moieties conjugated to the iRNA can optimize one or more properties of the iRNA. In many cases, this carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA can be linked to another moiety, for example, a non-carbohydrate (eg, cyclic) carrier to which a carbohydrate ligand is linked. The ribose sugars of the ribonucleotide subunits are referred to herein as ribose-modifying subunits (RRMS). The cyclic carrier may be a carbocyclic ring system, ie, all ring atoms are carbon atoms, or a heterocyclic ring system, ie, one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier may be a single ring system or may contain two or more rings, for example, fused rings. The cyclic carrier can be a fully saturated ring system, or it can contain one or more double bonds.

The ligand can be linked to the polynucleotide via a carrier. The carrier includes (i) at least one "backbone attachment point", such as two "backbone attachment points" and (ii) at least one "tethering attachment point". As used herein, "backbone attachment point" refers to a functional group, hydroxyl group, or generally a bond that is useful and suitable for incorporation of a carrier into a backbone, such as a phosphate or modified phosphate, for example, the sulfur-containing backbone of a ribonucleic acid. In some embodiments, a "tethering attachment point" (TAP) refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (different from the atoms that provide the backbone attachment point), which Connect a selected section. The moiety may be, for example, a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide. If necessary, selected parts can be The strap is connected to the loop carrier. Thus, cyclic carriers typically contain functional groups, such as amine groups, or generally provide bonds suitable for binding or binding another chemical entity, such as a ligand, to the constituent ring.

The iRNA can be connected to a ligand through a carrier, wherein the carrier can be a cyclic group or a non-cyclic group. In one embodiment, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]bis Oxopentane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl and decalinyl. In one embodiment, the acyclic group is a serinol skeleton or a diethanolamine skeleton.

i. Thermal destabilization modification

In some embodiments, RNA interference of dsRNA molecules can be optimized by incorporating thermal destabilizing modifications into the seed region of the antisense strand. As used herein, "seed region" refers to positions 2 to 9 at the 5'-end of the reference strand or positions 2 to 8 at the 5'-end of the reference strand. For example, heat-destabilizing modifications can be incorporated into the seed region of the antisense strand to reduce or inhibit off-target gene silencing.

The term "thermally destabilizing modification" includes one or more dsRNA modifications that will result in an overall demultiplexing temperature (Tm) that is lower than the Tm of a dsRNA that does not contain such modifications. For example, thermal destabilization modification can reduce the Tm of dsRNA by 1 to 4°C, such as 1, 2, 3 or 4°C. Furthermore, the term "thermally destabilizing nucleotide" refers to a nucleotide containing one or more thermally destabilizing modifications.

It has been found that dsRNA with an antisense strand containing at least one heat-labile modification of the duplex within the first 9 nucleotide positions of the antisense strand (counted from the 5’ end) has reduced detachment. Target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five or more ) Thermal labile modification of duplexes. In some embodiments, one or more thermal destabilizing modifications of the duplex are located at positions 2 to 9, such as positions 4 to 8, counting from the 5' end of the antisense strand. In some further embodiments, the heat-labile modification of the duplex is at position 6, 7, or 8 counted from the 5'-end of the antisense strand. In some further embodiments, the thermolabile modification of the duplex is located at position 7 of the 5'-end of the antisense strand. In some embodiments, the thermolabile modification of the duplex is located at position 2, 3, 4, 5, or 9 of the 5'-end of the antisense strand.

iRNA agents contain sense and antisense strands, each having 14 to 40 nucleotides. The RNAi agent can be represented by formula (L):

In formula (L), B1, B2, B3, B1', B2', B3' and B4' are independently selected from 2'-O-alkyl, 2'-substituted alkoxy modified nucleotides , the group consisting of 2'-substituted alkyl, 2'-halo, ENA and BNA/LNA. In one embodiment, B1, B2, B3, B1', B2', B3' and B4' all include 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3' and B4' all include 2'-OMe or 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3' and B4' contains 2'-O-N-methylacetyl group (2'-O-NMA, 2'O -CH2C(O)N(Me)H) modification.

C1 is a heat-labile nucleotide located at a position opposite the seed region of the antisense strand (i.e., position 28 at the 5'-end of the antisense strand or positions 2 to 9 at the 5'-end of the antisense strand) . For example, C1 is located at the position of the sense strand and pairs with nucleotides at positions 2 to 8 at the 5'-end of the antisense strand. In one embodiment, C1 is located at position 15 at the 5'-end of the sense strand. C1 nucleotides bear thermally labile modifications, which can include abasic modifications; mismatches with opposite nucleotides in the duplex; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, For example, unlocked nucleic acid (UNA), glycerol nucleic acid (GNA), or 2'-5'-linked ribonucleotides ("3'-RNA"). In one embodiment, C1 has a thermally labile modification selected from the group consisting of: i) a mismatch with the opposite nucleotide in the antisense strand; ii) an abasic modification selected from the group consisting of: Modification:

；iii)選自由以下組成的 群組的糖修飾：

;iii) Sugar modification selected from the group consisting of:

，其中B係修飾或未修飾的核鹼基，R¹和R²獨立係H、鹵 素、OR₃或烷基；R₃係H、烷基、環烷基、芳基、芳烷基、雜芳基或糖。在一實施態樣中，C1中的熱不穩定修飾係選自由G：G、G：A、G：U、 G：T、A：A、A：C、C：C、C組成的組的錯配：U、C：T、U：U、T：T和U：T所組成之群組；並且視需要地，錯配對中的至少一核鹼基係2'-去氧核鹼基。在一實施例中，該C1中的熱不穩定化修飾係GNA或

, where B is a modified or unmodified nucleobase, R ¹ and R ² are independently H, halogen, OR ₃ or alkyl; R ₃ is H, alkyl, cycloalkyl, aryl, aralkyl, hetero Aryl or sugar. In an embodiment, the thermally unstable modification in C1 is selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, and C. Mismatch: the group consisting of U, C:T, U:U, T:T and U:T; and optionally, at least one nucleobase in the mismatch is a 2'-deoxynucleobase. In one embodiment, the thermally unstable modification in C1 is GNA or

T1, T1', T2', and T3' each independently represent a nucleotide that contains a modification that provides a steric volume less than or equal to the steric volume of the 2'-OMe modification. Spatial volume refers to the sum of the spatial effects of modifications. Methods for determining the steric effects of nucleotide modifications are known to those of ordinary skill in the art. The modification can be at the 2' position of the ribose sugar of the nucleotide, or a modification of a non-ribonucleotide, acyclic nucleotide, or a nucleotide backbone that is similar or equivalent to the 2' position of the ribose sugar, and is provided by the nucleotide A volume of space less than or equal to the volume of space modified by 2'-OMe. For example, T1, T1', T2' and T3' are each independently selected from DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA.

n ¹ , n ³ and q ¹ are independently 4 to 15 nucleotides in length.

n ⁵ , q ³ and q ⁷ are independently 1 to 6 nucleotides in length.

n ⁴ , q ² and q ⁶ are independently 1 to 3 nucleotides in length; alternatively, n ⁴ is 0.

q ⁵ independently ranges from 0 to 10 nucleotides in length.

n ² and q ⁴ are independently 0 to 3 nucleotides in length.

Alternatively, n ⁴ is from 0 to 3 nucleotides in length.

In one implementation, n ⁴ can be zero. In one embodiment, n ⁴ is 0, and q ² and q ⁶ are 1. In another embodiment, n ⁴ is 0, and q ² and q ⁶ are 1, with two phosphorothioate nucleosides within sense strand positions 1 to 5 (counting from the 5'-end of the sense strand) Interacid linkage modification, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 of the antisense strand Chain link modifications (counted from the 5'-end of the antisense strand).

In one implementation, each of n ⁴ , q ² and q ⁶ is 1.

In one implementation, each of n ² , n ⁴ , q ² , q ⁴ and q ⁶ is 1.

In one embodiment, when the sense strand is 19 to 22 nucleotides in length and n ⁴ is 1, C1 is located at positions 14 to 17 at the 5'-end of the sense strand. In one embodiment, C1 is located at position 15 at the 5'-end of the sense strand.

In one embodiment, T3' begins at position 2 at the 5' end of the antisense strand. In one embodiment, T3' is located at position 2 at the 5' end of the antisense strand, and ^q6 equals one.

In one embodiment, T1' begins at position 14 at the 5' end of the antisense strand. In one embodiment, T1' is located at position 14 at the 5' end of the antisense strand, and ^q2 equals one.

In one exemplary embodiment, T3' starts at position 2 at the 5' end of the antisense strand, and T1' starts at position 14 at the 5' end of the antisense strand. In one embodiment, T3' starts from position 2 at the 5' end of the antisense strand, and q ⁶ is equal to 1, and T1' starts from position 14 at the 5' end of the antisense strand, and q ² is equal to 1.

In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, excluding the nucleotides of T1' and T3').

In one embodiment, T1' is located at position 14 at the 5' end of the antisense strand. In one embodiment, T1' is at position 14 at the 5' end of the antisense strand, and ^q2 equals 1, and modification at position 2' or modifications at multiple positions in the non-ribose, acyclic, or backbone provides less than The spatial volume of 2'-OMe ribose.

In one embodiment, T3' is located at position 2 at the 5' end of the antisense strand. In one embodiment, T3' is located at position 2 of the 5' end of the antisense strand, and q equals ¹ , and modification at position 2' or at multiple positions in the non-ribose, acyclic, or backbone provides less Is equal to or equal to the spatial volume of 2'-OMe ribose.

In one embodiment, T1 is located at the cleavage site of the sense strand. In one embodiment, when the length of the sense strand is 19 to 22 nucleotides and n ² is 1, T1 is located at position 11 at the 5' end of the sense strand. In an exemplary embodiment, when the length of the sense strand is 19 to 22 nucleotides and ⁿ² is 1, T1 is located at the cleavage site at position 11 at the 5' end of the sense strand.

In one embodiment, T2' begins at position 6 at the 5' end of the antisense strand. In one embodiment, T2' is located at positions 6 to 10 at the 5' end of the antisense strand, and ^q4 is 1.

In an exemplary embodiment, T1 is at the cleavage site of the sense strand, e.g., at position 11 at the 5' end of the sense strand, when the sense strand is 19 to 22 nucleotides in length and n ² is 1; T1' is located at position 14 at the 5' end of the antisense strand, and q ² is equal to 1, and the modification to T1' is located at the 2' position of ribose or at multiple positions in non-ribose, acyclic or backbone Provides less space than 2'-OMe ribose; T2' is located at position 6 to 10 at the 5' end of the antisense strand, q ⁴ is 1; and T3' is located at position 2 at the 5' end of the antisense strand, q ⁶ is equal to 1 , modifications to T3' are located at position 2' or are non-ribose, acyclic, or modifications at multiple positions in the backbone provide less than or equal space volume to 2'-OMe ribose.

In one embodiment, T2' begins at position 8 at the 5' end of the antisense strand. In one embodiment, T2' begins at position 8 at the 5' end of the antisense strand, and ^q4 is 2.

In one embodiment, T2' begins at position 9 at the 5' end of the antisense strand. In one embodiment, T2' is located at position 9 at the 5' end of the antisense strand, and ^q4 is 1.

In one embodiment, B1' is 2'-OMe or 2'-F, ^q1 is 9, T1' is 2'-F, ^q2 is 1, and B2' is 2'-OMe or 2'-F. , q ³ is 4, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2'-F, q ⁵ is 6, T3' is 2'-F, q ⁶ is 1, B4' is 2'-OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand) , and there are two phosphorothioate internucleotide link modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide link modifications at positions 18 to 23 (from the antisense strand 5'-end count).

In one embodiment, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, and T1' is 2'-F. q ² series 1, B2' series 2'-OMe or 2'-F, q ³ series 4, T2' series 2'-F, q ⁴ series 1, B3' series 2'-OMe or 2'-F, q ⁵ series 6, T3' series 2'-F, q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate nucleotides in positions 1 to 5 of the sense strand Inter-linkage modifications (counted from the 5'-end of the sense strand), and two phosphorothioate inter-linkage modifications at positions 1 and 2 of the antisense strand and two between positions 18 to 23 Phosphorothioate internucleotide link modification (counted from the 5'-end of the antisense strand).

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-OMe, q ⁷ is 1.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand).

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 6, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 7, T1' is 2'-F, q2 is 1, B2' is 2'-OMe Or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F, q ⁶ is series 1, B4' is 2'-OMe, q ⁷ is series 1.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 6, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 7, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand).

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2'-F, q ⁵ is 6, T3' is 2'-F , q ⁶ is 1, B4' is 2'-OMe, q ⁷ is 1.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2'-F, q ⁵ is 6, T3' is 2'-F , q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5' of the sense strand - end count), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 ( Counting from the 5'-end of the antisense strand).

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 5, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; if necessary, have at least 2 additional TTs in the 3'-end of the antisense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 5, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; optionally, at least 2 additional TTs in the 3'-end of the antisense strand; within positions 1 to 5 of the sense strand Two phosphorothioate internucleotide linkage modifications (counted from the 5'-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counted from the 5'-end of the antisense strand).

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2 'OMe, q ⁷ is 1.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'OMe, q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counting from the 5'-end), and at positions 1 to 5 of the antisense strand 1 and 2 have two phosphorothioate internucleotide linkage modifications and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counted from the 5'-end).

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-F, q ⁷ is 1.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ series 1, B4' series 2'-F, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5' of the sense strand -end count), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 ( Counting from the 5'-end of the antisense strand).

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, q ⁷ is 1.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, ^q7 is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in the reverse The sense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the 5'- end count).

)。當5'-末端含磷基 團係5'-末端乙烯基膦酸酯(5'-VP)時，該5'-VP可以係5'-E-VP異構體(即， 反式-乙烯基膦酸酯，

)、5'-Z-VP異構體(即，順式-乙烯基膦酸 酯，

)或其混合物。 The RNAi agent may contain a phosphorus-containing group at the 5'-end of the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-terminal -PS ₂ ), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos) or 5'-deoxy-5'-C-malonylphosphonate (

). When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP can be a 5'-E-VP isomer (i.e., trans-ethylene base phosphonate,

), 5'-Z-VP isomer (i.e., cis-vinylphosphonate,

) or mixtures thereof.

In one embodiment, the RNAi agent contains a phosphorus-containing group at the 5'-end of the sense strand. In one embodiment, the RNAi agent includes a phosphorus-containing group at the 5'-end of the antisense strand.

In one embodiment, the RNAi agent includes 5'-P. In one embodiment, the RNAi agent includes 5'-P in the antisense strand.

In one embodiment, the RNAi agent includes 5'-PS. In one embodiment, the RNAi agent includes 5'-PS in the antisense strand.

In one embodiment, the RNAi agent includes 5'-VP. In one embodiment, the RNAi agent includes 5'-VP in the antisense strand. In one embodiment, the RNAi agent includes 5'- E -VP in the antisense strand. In one embodiment, the RNAi agent includes 5'- Z -VP in the antisense strand.

In one embodiment, the RNAi agent includes 5'- _PS2 . In one embodiment, the RNAi agent includes 5'- _PS2 in the antisense strand.

In one embodiment, the RNAi agent includes 5'- _PS2 . In one embodiment, the RNAi agent includes a 5'-deoxy-5'-C-malonyl group in the antisense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-OMe, q ⁷ is 1. The RNAi agent also contains 5'-PS.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-OMe, and q ⁷ is 1. The RNAi agent also contains 5'-P.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-OMe, q ⁷ is 1. The RNAi agent also contains a 5'-VP. The 5'-VP can be 5'- E -VP, 5'- Z -VP or a combination thereof.

In one embodiment, B1 is 2'-OMeor 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'-OMe or 2'-F, q ³ series 4, T2' series 2'-F, q ⁴ series 2, B3' series 2'-OMe or 2'-F, q ⁵ series 5, T3' series 2'-F, q ⁶ is 1, B4' is 2'-OMe and q ⁷ is 1. The RNAi agent also contains 5'- _PS2 .

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-OMe, q ⁷ is 1. The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-P.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-PS.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-VP. 5'-VP can be 5'- E -VP, 5'- Z -VP or a combination thereof.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'- _PS2 .

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-OMe, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q5 series 7, T3' series 2'-F, q6 series 1, B4' series 2'OMe, q7 is 1. The RNAi agent also contains 5'-P.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9. T1' is 2'-F, q ² is 1, B2' is 2'-OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2'-F, q ⁵ is 7, T3' is 2'-F, q ⁶ is 1, B4' is 2'OMe, q ⁷ is 1. The dsRNA agent also contains 5'-PS.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2 'OMe, q ⁷ is 1. The RNAi agent also contains 5'-VP. The 5'-VP can be 5'- E -VP, 5'- Z -VP or a combination thereof.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2 'OMe, q ⁷ is 1. This RNAi agent also contains 5'-PS ₂

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2 'OMe, q ⁷ is 1. The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'OMe, q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counting from the 5'-end), and at positions 1 to 5 of the antisense strand 1 and 2 have two phosphorothioate internucleotide linkage modifications and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains 5'-P.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'OMe, q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counting from the 5'-end), and at positions 1 to 5 of the antisense strand 1 and 2 have two phosphorothioate internucleotide linkage modifications and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains 5'-PS.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B ³ is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2 '-OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2'-F, q ⁵ is 7, T3' is 2'-F, q ⁶ is 1, B4' is 2'OMe, ^q7 is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counted from the 5'-end), and in the antisense strand There are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains 5'-VP. The 5'-VP can be 5'- E -VP, 5'- Z -VP or a combination thereof.

In one embodiment, B1 is 2'-OMe or 2'-F, ⁿ¹ is 8, T1 is 2'F, n2 is 3, B2 is 2'-OMe, n3 is 7, n4 is 0, and B3 The system is 2'-OMe, n5 is 3, B1' is 2'-OMe or 2'-F, q1 is 9, T1' is 2'-F, q2 is 1, B2' is 2'-OMe or 2'- F, q3 is 4, q4 is 0, B3' is 2'-OMe or 2'-F, q5 is 7, T3' is 2'-F, q6 is 1, B4' is 2'OMe, q7 is 1; There are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counted from the 5'-end), and two phosphorothioate cores in positions 1 and 2 of the antisense strand Internucleotide linkage modifications and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains 5'- _PS2 .

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B ³ is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2 '-OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2'-F, q ⁵ is 7, T3' is 2'-F, q ⁶ is 1, B4' is 2'OMe, ^q7 is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counted from the 5'-end), and in the antisense strand There are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , ^q6 is 1, B4' is 2'-F, q7 is 1. The RNAi agent also contains 5'-P.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , ^q6 is 1, B4' is 2'-F, q7 is 1. The RNAi agent also contains 5'-PS.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-F, q ⁷ is 1. The RNAi agent also contains 5'-VP. 5'-VP can be 5'- E -VP, 5'- Z -VP or a combination thereof.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-F, q ⁷ is 1. The dsRNAiRNA agent also contains 5'- _PS2 .

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'- OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'-F , q ⁶ is 1, B4' is 2'-F, and q ⁷ is 1. The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-F, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-P.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-F, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-PS.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-F, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-VP. The 5'-VP can be 5'- E -VP, 5'- Z -VP or a combination thereof.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-F, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'- _PS2 .

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ series 1, B4' series 2'-F, q ⁷ series 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B ³ is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2 '-OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2'-F, q ⁵ is 7, T3' is 2'-F, q ⁶ is 1, B4' is 2'F, q ⁷ is 1. The RNAi agent also contains 5'-P.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, q ⁷ is 1. The RNAi agent also contains 5'-PS.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, q ⁷ is 1. The RNAi agent also contains 5'-VP. The 5'-VP can be 5'- E -VP, 5'- Z -VP or a combination thereof.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, q ⁷ is 1. The RNAi agent also contains 5'- _PS2 .

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, q ⁷ is 1. The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, ^q7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in the reverse The sense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the 5'- end count). The RNAi agent also contains 5'-P.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, ^q7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in the reverse The sense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the 5'- end count). The RNAi agent also contains 5'-PS.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, ^q7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in the reverse The sense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the 5'- end count). The RNAi agent also contains 5'-VP. The 5'-VP can be 5'- E -VP, 5'- Z -VP or a combination thereof.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, ^q7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in the reverse The sense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the 5'- end count). The RNAi agent also contains 5'- _PS2 .

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'F, ^q7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in the reverse The sense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the 5'- end count). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

In one embodiment, B1 is 2'-OMeor 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'-OMeor2 '-F, q ³ series 4, T2' series 2'-F, q ⁴ series 2, B3' series 2'-OMeor2'-F, q ⁵ series 5, T3' series 2'-F, q ⁶ series 1 , B4' is 2'-OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand) , and there are two phosphorothioate internucleotide link modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide link modifications at positions 18 to 23 (from the antisense strand 5'-end count). The RNAi agent also contains 5'-P and targeting ligands. In one embodiment, the 5'-P is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'-OMeor2'-F, q ³ series 4, T2' series 2'-F, q ⁴ series 2, B3' series 2'-OMeor2'-F, q ⁵ series 5, T3' series 2'-F, q ⁶ Line 1, B4' line 2'-OMe, and q ⁷ line 1; two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (from the 5'-end of the sense strand count), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the antisense strand 5'-end count of the sense stock). The RNAi agent also contains 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'-OMeor2'-F, q ³ series 4, T2' series 2'-F, q ⁴ series 2, B3' series 2'-OMeor2'-F, q ⁵ series 5, T3' series 2'-F, q ⁶ Line 1, B4' line 2'-OMe, and q ⁷ line 1; two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (from the 5'-end of the sense strand count), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the antisense strand 5'-end count of the sense stock). The RNAi agent also contains a5'-PS and a targeting ligand. The RNAi agent also includes a 5'-VP (eg, 5'- E -VP, 5'- Z -VP, or a combination thereof), and a targeting ligand.

In one embodiment, the 5'-VP is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'-OMeor2'-F, q ³ series 4, T2' series 2'-F, q ⁴ series 2, B3' series 2'-OMeor2'-F, q ⁵ series 5, T3' series 2'-F, q ⁶ Line 1, B4' line 2'-OMe, and q ⁷ line 1; two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (from the 5'-end of the sense strand count), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the antisense strand 5'-end count of the sense stock). The RNAi agent also contains 5'-PS ₂ and a targeting ligand. In one embodiment, the 5'-PS ₂ is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'-OMeor2'-F, q ³ series 4, T2' series 2'-F, q ⁴ series 2, B3' series 2'-OMeor2'-F, q ⁵ series 5, T3' series 2'-F, q ⁶ Line 1, B4' line 2'-OMe, and q ⁷ line 1; two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (from the 5'-end of the sense strand count), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from the antisense strand 5'-end count of the sense stock). The RNAi agent also contains a 5'-deoxy-5'- C -malonyl group and a targeting ligand. In one embodiment, the 5'-deoxy-5'- C -malonyl group is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'-OMeor2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMeor2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4' series 2' OMe, and q ⁷ series 1; two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counting from the 5'-end), and positions 1 and 2 of the antisense strand There are two phosphorothioate internucleotide linkage modifications and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains 5'-P and targeting ligands. In one embodiment, the 5'-P is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'-OMe, and ^q7 is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counting from the 5'-end), and in the antisense strand There are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'-OMe, and ^q7 is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counting from the 5'-end), and in the antisense strand There are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (counted from the 5'-end). The RNAi agent also includes a 5'-VP (eg, 5'- E -VP, 5'- Z -VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'OMe, and q7 is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 on the sense strand (counting from the 5'-end), and at positions 1 to 5 on the antisense strand 1 and 2 have two phosphorothioate internucleotide linkage modifications and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains 5'-PS ₂ and a targeting ligand. In one embodiment, the 5'-PS ₂ is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMeor 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2'-OMe Or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4' series 2'-OMe, and q ⁷ series 1; two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counting from the 5'-end), and in positions 1 to 5 of the antisense strand 1 and 2 have two phosphorothioate internucleotide linkage modifications and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counted from the 5'-end). The RNAi agent also contains a 5'-deoxy-5'- C -malonyl group and a targeting ligand. In one embodiment, the 5'-deoxy-5'- C -malonyl group is at the 5'-end of the antisense strand, and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ is 1, B4' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand) '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-P and targeting ligands. In one embodiment, the 5'-P is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ is 1, B4' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand) '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ is 1, B4' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand) '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also includes a 5'-VP ( eg , 5'- E -VP, 5'- Z -VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ is 1, B4' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications within positions 1 to 5 of the sense strand (from 5 of the sense strand) '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains 5'-PS ₂ and a targeting ligand. In one embodiment, the 5'-PS ₂ is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2'-F, q ⁵ is 5, T3' is 2'- F, q ⁶ is 1, B4' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (from 5 of the sense strand) '-end count), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (Counted from the 5'-end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'- C -malonyl group and a targeting ligand. In one embodiment, the 5'-deoxy-5'- C -malonyl group is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications within positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in The antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from 5' of the antisense strand -end count). The RNAi agent also contains 5'-P and targeting ligands. In one embodiment, the 5'-P is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications within positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in The antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from 5' of the antisense strand -end count). The RNAi agent also contains 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications in positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in The antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from 5' of the antisense strand -end count). The RNAi agent also includes a 5'-VP ( eg , 5'- E -VP, 5'- Z -VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications within positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in The antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from 5' of the antisense strand -end count). The RNAi agent also contains 5'-PS ₂ and a targeting ligand. In one embodiment, the 5'-PS ₂ is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In one embodiment, B1 is 2'-OMe or 2'-F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2'-OMe, n ³ is 7, and n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2'-F, q ¹ is 9, T1' is 2'-F, q ² is 1, B2' is 2' -OMe or 2'-F, q ³ series 4, q ⁴ series 0, B3' series 2'-OMe or 2'-F, q ⁵ series 7, T3' series 2'-F, q ⁶ series 1, B4 ' is 2'-F and q ⁷ is 1; there are two phosphorothioate internucleotide link modifications within positions 1 to 5 of the sense strand (counted from the 5'-end of the sense strand), and in The antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 18 to 23 (from 5' of the antisense strand -end count). The RNAi agent also contains a 5'-deoxy-5'- C -malonyl group and a targeting ligand. In one embodiment, the 5'-deoxy-5'- C -malonyl group is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

In a specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand linked to the 3′-terminus, wherein the ASGPR ligand includes three GalNAc derivatives attached by a trivalent branched linker; and

(iii) 2,-F modification at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19 and 21 and at positions 2, 4, 6, 8, 12, 14 to 16, 18 and 20 2'-OMe modification (counted from the 5' end);

and

(b) Antisense stocks contain:

(i) 23 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21 and 23 and at positions 2, 4, 6 to 8, 10, 14, 16, 18 , 2'F modification of 20 and 22 (counted from the 5' end); and

(iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counted from the 5’ end);

The dsRNA agent contains a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand connected to the 3'-terminus, wherein the ASGPR ligand comprises three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19 and 21 and at positions 2, 4, 6, 8, 12, 14, 16, 18 and 20 2'-OMe modifications (counted from the 5' end); and

(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5’ end);

and

(b) Antisense stocks contain:

(i) 23 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19 and 21 to 23 and at positions 2, 4, 6, 8, 10, 14, 16 2'F modification of , 18, and 20 (counted from the 5' end);

as well as

(iii) Phosphorothioate nucleotides between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 Interlinker (counted from 5' end);

wherein the RNAi agent contains a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand connected to the 3'-terminus, wherein the ASGPR ligand comprises three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2'-OMe modifications at positions 1 to 6, 8, 10 and 12 to 21 and 2'-F modifications at positions 7 and 9 and a deoxynucleotide (e.g. dT) at position 11 (from 5' end count); and

(iv) phosphorothioate internucleotide links between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5’ end);

as well as

(b) Antisense stocks contain:

(i) 23 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17 and 19 to 23 and at positions 2, 4 to 6, 8, 10, 12, 14, 16 and 18 2'-F modification (counted from the 5' end); and

(iii) Phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 Internucleotide links (counted from 5' end);

The RNAi agent contains a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand connected to the 3'-terminus, wherein the ASGPR ligand comprises three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2’-OMe modifications at positions 1 to 6, 8, 10, 12, 14 and 16 to 21 and 2’-F modifications at positions 7, 9, 11, 13 and 15; and

(iv) a phosphorothioate internucleotide link between nucleotide positions 1 and 2 and nucleotide positions 2 and 3 (counted from the 5’ end);

as well as

(b) Antisense stocks contain:

(i) 23 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19 and 21 to 23 and at positions 2 to 4, 6, 8, 10, 12, 14, 16 , 2'-F modification between 18 and 20 (counted from the 5' end); and

(iii) Phosphorothioate nucleosides between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 interacid linkage (counted from 5' end);

The RNAi agent contains a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand connected to the 3'-terminus, wherein the ASGPR ligand comprises three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2’-OMe modification at positions 1 to 9 and 12 to 21 and 2’-F modification at positions 10 and 11; and

(iv) phosphorothioate internucleotide links at nucleotide positions 1 and 2 and nucleotide positions 2 to 3 (counted from the 5′ end);

as well as

(b) Antisense stocks contain:

(i) 23 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19 and 21 to 23 and at positions 2, 4, 6, 8, 10, 14, 16 2'-F modification of , 18 and 20 (calculated from the 5' end);

as well as

(iii) Sulfur between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (calculated from the 5' end) phosphoric acid ester internucleotide linkage;

The RNAi agent contains a 2-nucleotide protrusion at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand connected to the 3'-terminus, wherein the ASGPR ligand comprises three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11 and 13 and 2'-OMe modifications at positions 2, 4, 6, 8, 12 and 14 to 21 (from 5' end count); and

(iv) phosphorothioate internucleotide links between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5’ end);

as well as

(b) Antisense stocks contain:

(i) 23 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19 and 21 to 23 and at positions 2, 4, 8, 10, 14, 16 and 202 '-F modification (counted from the 5' end); and

(iii) Phosphorothioate internucleotide strands between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 Knots (counted from 5' end);

The RNAi agent contains a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand connected to the 3'-terminus, wherein the ASGPR ligand comprises three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19 to 21 and 2'-OMe modifications at positions 3, 5, 7, 9 to 11, 13, 16, and 2'-F modification of 18; and

(iv) phosphorothioate internucleotide linkages at nucleotide positions 1 and 2, and at nucleotide positions 2 and 3 (counting from the 5′ end);

as well as

(b) Antisense stocks contain:

(i) 25 nucleotides in length;

(ii) 2'-OMe modification at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17 and 19 to 23, 2'-OMe modification at positions 2, 3, 5, 8, 10, 14, 16 and 18 2'-F modification, and deoxynucleotides (e.g., dT) at positions 24 and 25 (counted from the 5' end); and

(iii) Phosphorothioate nucleotides between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 Interlinker (counted from 5' end);

The RNAi agent contains a 4-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand connected to the 3′-terminus, wherein the ASGPR ligand includes three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2’-OMe modification at positions 1 to 6, 8 and 12 to 21 and 2’-F modification at positions 7 and 9 to 11; and

(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5’ end);

as well as

(b) Antisense stocks contain:

(i) 23 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15 and 17 to 23 and 2'-F modifications at positions 2, 6, 9, 14 and 16 (from 5' end count); and

(iii) Phosphorothioate nucleotides between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 Interlinker (counted from 5' end);

The RNAi agent contains a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the present disclosure includes:

(a) Equity shares include:

(i) 21 nucleotides in length;

(ii) an ASGPR ligand connected to the 3'-terminus, wherein the ASGPR ligand comprises three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2’-OMe modification at positions 1 to 6, 8 and 12 to 21 and 2’-F modification at positions 7 and 9 to 11; and

(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5’ end);

as well as

(b) Antisense stocks contain:

(i) 23 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15 and 17 to 23 and 2'-F modifications at positions 2, 6, 8, 9, 14 and 16 (from 5' end count); and

(iii) Phosphorothioate nucleosides between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 interacid linkage (counted from 5' end);

The RNAi agent contains a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In another specific embodiment, the RNAi agent of the invention includes:

(a) Equity shares include:

(i) 19 nucleotides in length;

(ii) an ASGPR ligand connected to the 3'-terminus, wherein the ASGPR ligand comprises three GalNAc derivatives attached by a trivalent branched linker;

(iii) 2’-OMe modification at positions 1 to 4, 6 and 10 to 19 and 2’-F modification at positions 5 and 7 to 9; and

(iv) phosphorothioate internucleotide links between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5’ end);

as well as

(b) Antisense stocks contain:

(i) 21 nucleotides in length;

(ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15 and 17 to 21 and 2'-F modifications at positions 2, 6, 8, 9, 14 and 16 (from 5' end count); and

(iii) Between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 phosphorothioate internucleotides Links (counted from 5' end);

The RNAi agent contains a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

In some embodiments, the agent for iRNA used in the methods of the present disclosure is selected from any of the agents listed in Tables 2-7. These agents may also contain ligands.

III. iRNAs that engage ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, For example, enter a cell. These moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al. , Proc. Natl. Acid. Sci. USA , 1989, 86: 6553-6556). In other embodiments, the ligand is cholic acid (Manoharan et al. , Biog. Med. Chem. Let. , 1994, 4: 1053-1060), thioether, for example, hexyl-S-trityl Thiols (Manoharan et al. , Ann. NYAcad. Sci. , 1992, 660: 306-309; Manoharan et al. , Biog. Med. Chem. Let. , 1993, 3: 2765-2770), thiocholesterol (Oberhauser et al. , Nucl. Acids Res. , 1992, 20: 533-538), aliphatic chains, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al. , EMBO J , 1991,10:1111-1118; Kabanov et al. , FEBS Lett. , 1990,259:327-330; Svinarchuk et al. , Biochimie , 1993,75:49-54), phospholipids, for example, hexadecyl - Racemic glycerol or triethylammonium 1,2-di-O-hexadecyl-racemic glycerol-3-phosphonate (Manoharan et al. , Tetrahedron Lett. , 1995, 36: 3651-3654 ; Shea et al. , Nucl. Acids Res. , 1990, 18: 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al. , Nucleosides & Nucleotides , 1995, 14: 969-973) or adamantane Acetic acid (Manoharan et al. , Tetrahedron Lett. , 1995, 36: 3651-3654), palmyl moiety (Mishra et al. , Biochim. Biophys. Acta , 1995, 1264: 229-237) or stearylamine or hexylamine -carbonyloxycholesterol moiety (Crooke et al. , J. Pharmacol. Exp. Ther. , 1996, 277: 923-937).

In some embodiments, a ligand alters the distribution, targeting, or lifetime of the iRNA agent into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target, e.g., a molecule, cell or cell type, compartment, e.g., a cell or organ compartment, tissue, organ or region of the body, as, for example, compared to species without such a coordination partner. In some embodiments, the ligand does not participate in duplex-to-duplex and nucleotide pairing.

Ligands may include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), or globulin); carbohydrates (e.g., glucans, pullulan, chitin substance, chitosan, inulin, cyclodextrin, N-acetylglucose, N-acetylgalactosamine or hyaluronic acid); or lipid. The ligand can be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include polyamino acid-based polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide) Ester-co-ethylene glycol) copolymer, divinyl ether-malic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), poly Vinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer or polyphosphazine. Examples of polyamino acids include: polyethylenimine, polylysine acid (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendritic polyamines, spermine Amino acids, amidines, protamine, cationic lipids, cationic porphyrins, the third salt of polyamines or α-helical peptides.

Ligands may include targeting groups, eg, agents that target cells or tissues, eg, lectins, glycoproteins, lipids, or proteins, eg, antibodies that bind to specific cell types, such as kidney cells. Targeting groups can be thyrotropin, melanogen, lectin, glycoprotein, surface activity Protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosyl Polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin Or RGD peptide or RGD peptide mimetic. In some embodiments, the ligand is a polyvalent galactose, for example, N-acetyl-galactosamine.

Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, saphirin) (Sapphyrin)), polycyclic aromatic hydrocarbons (such as phenazine, dihydrophenazine), artificial endonucleases (such as EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, Dihydrotestosterone, 1,3-bis-O(cetyl)glycerol, geranyloxyhexyl, cetylglycerin, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, Myristic acid, O3-(oleyl)lithocholic acid, O3-(oleyl)cholic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide , Tat peptide), alkylating agent, phosphate, amine, thiol, PEG (such as PEG-40K), MPEG, [MPEG]2, polyamine, alkyl, substituted alkyl, radiolabeled label , enzymes, haptens (e.g., biotin), transport/absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histidine, imidazole cluster, acridine-imidazole Conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

The ligand may be a protein, such as a glycoprotein, or a peptide, eg, a molecule with a specific affinity for the coligand or antibody, eg, an antibody that binds to a specific cell type, such as hepatocytes. Ligands may also include hormones and hormone receptors. They may also include non-peptide substances such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent half- Lactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose or polyvalent fucose. The ligand may be, for example, lipopolysaccharide (an activator of p38 MAP kinase) or an activator of NF-κB.

The ligand can be a substance, eg, a drug, that increases the uptake of the iRNA agent into the cell, eg, by disrupting the cell's cytoskeleton, eg, by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug may be, for example, paclitaxel, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, red sea spongin A, phalloidin, swinholide A, Indanocine or myoservin.

In some embodiments, a ligand associated with an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilicity, cholic acid, steroids, phospholipid analogs, peptides, protein binding agents, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biologic white. Oligonucleotides containing many phosphorothioate links are known to bind to serum proteins, so short oligonucleotides containing many phosphorothioate links in the backbone, e.g., 5 bases, 10 bases , 15 bases or 20 bases as ligands are also suitable for use in this disclosure (eg, as PK modulating ligands). Additionally, aptamers that bind serum components (eg, serum proteins) are also suitable for use as PK modulating ligands in embodiments described herein.

Ligand-joining iRNAs of the present invention can be synthesized by using oligonucleotides with pendant reactive functions, such as those derived from linker molecules attached to the oligonucleotides (as described below). The reaction oligonucleotide can be reacted directly with commercially available ligands, synthetic ligands bearing various protecting groups, or ligands having moieties attached thereto.

The oligonucleotides used for conjugation of the present invention can be conveniently and fixedly prepared by well-known solid-state synthesis techniques. Equipment for such synthesis is sold by many manufacturers, such as Applied Biosystems® (Foster City, Calif.). Any other method known in the art for such synthesis may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

In ligand-conjugated iRNAs and ligand molecules containing sequence-specific nucleosides of the invention, the oligonucleotides and oligonucleotides may utilize standard nucleotide or nucleoside precursors, or may have The nucleotide or nucleoside conjugation precursor of the linking portion, the ligand nucleotide or nucleoside conjugation precursor that already bears the ligand molecule, or the structural unit of the non-nucleotide conjugation ligand, in the appropriate Assembled on a DNA synthesizer.

Synthesis of the sequence-specific linking nucleoside is typically accomplished when using a nucleotide conjugation precursor that already carries a linker moiety, and the ligand moiety is an oligonucleotide that reacts with the linker moiety to form a conjugate ligand. . In some embodiments, oligonucleotides or linked nucleosides of the present disclosure are synthesized by automated synthesizers using phosphoramidites derived from ligand nucleoside conjugates and commercially available and commonly used Standard and non-standard phosphoramidites for oligonucleotide synthesis.

A. Lipid conjugates

In some embodiments, the antibody or conjugate is a lipid or lipid-based molecule. In one embodiment, the lipid or lipid-based molecule binds to a serum protein, such as human serum albumin (HSA). HSA bound to a ligand allows distribution of the conjugate to target tissues, for example, non-kidney target tissues of the body. For example, the target tissue may be liver, including liver parenchymal cells. Other molecules that can bind to HSA can also serve as ligands. For example, naproxen or aspirin may be used. Lipids or lipid-based molecules may (a) increase conjugate degradation resistance, (b) increase targeting or translocation into target cells or cell membranes, or (c) can be used to modulate binding to serum proteins, e.g., HSA.

Lipid-based ligands can be used to inhibit, for example, control the binding of the conjugate to the target tissue. For example, lipids or lipid-based ligands that bind to HSA are less likely to be targeted to the kidneys and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind to HSA can be used to target engagement with the kidney.

In some embodiments, a lipid-based ligand binds to HSA. In one embodiment, it binds to HSA with sufficient affinity such that the conjugate will distribute to non-kidney tissue. Preferably, however, the affinity is not so strong that it cannot reverse HSA ligand binding.

In other embodiments, the lipid-based ligand binds HSA weakly or not at all. In one embodiment, the conjugate will be distributed to the kidneys. Targeting other moieties of kidney cells may also be used instead of or in addition to lipid-based ligands.

In another aspect, the ligand, eg, a vitamin, is a moiety that is taken up by the target cell, eg, a proliferating cell. These are particularly suitable for the treatment of disorders characterized by undesirable cell proliferation, eg of malignant or non-malignant types, eg cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins such as folate, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients absorbed by target cells such as hepatocytes. Also includes HSA and low-density lipoprotein (LDL).

B. Cell penetrant

In another aspect, the ligand is a cell penetrant, such as a helix cell penetrant. In one embodiment, the agent is amphipathic. Exemplary agents are peptides such as tat or ant. like If the agent is a peptide, it may be modified, including peptide mimetics, transformants, non-peptide or pseudopeptide links, and the use of D-amino acids. In one embodiment, the spiral agent is an alpha spiral agent, which has a lipophilic phase and a lipophobic phase.

The ligand can be a peptide or a peptide mimetic. Peptide mimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into a 3D structure similar to natural peptides. The addition of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic profile of the iRNA, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion may be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length.

The peptide or peptidomimetic may be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (eg, consisting primarily of Tyr, Trp or Phe). The peptide moiety may be a dendritic peptide, a constrained peptide, or a cross-linked peptide. In another option, the peptide moiety may include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF containing the amino acid series AAVALLPAVLLALLAP (SEQ ID NO: 14). RFGF isomers (e.g., the hydrophobic MTS-containing amino acid sequence AALLPVLLAAP (SEQ ID NO: 15)) can also be a targeting moiety. The peptide moiety can be a "transport" peptide, which can carry large amounts of polar molecules Pass through the cell membrane, including peptides, oligonucleotides and proteins. For example, it has been found that the sequences from HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 16) and Drosophila antennal protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 17)) can exert Such as the function of transporting peptides. Peptides or peptoids can be encoded by random sequences of DNA, such as identified from phage display databases or one-bead-one-compound (OBOC) combinatorial databases (Lam et al., Nature, 354 : 82-84, 1991). An example of a peptide or peptoid linked to a dsRNA agent by incorporated monomeric units for cell targeting purposes is Arginine-Glycine-Aspartate (RGD)-win Peptide or RGD mimetics. The peptide portion can be from about 5 amino acids to about 40 amino acids in length. The peptide moiety may have structural modifications, such as properties that increase stability or guide conformation. Any of the structural modifications described below may be used.

RGD peptides useful in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues. RGD-containing peptides and peptidomimetics may include D-amino acids as well as synthetic RGD mimetics. In addition to RGD, other moieties targeting integrin ligands can also be used, for example, PECAM-1 or VEGF.

Cell-penetrating peptide systems can penetrate cells, for example, microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Peptides that penetrate microbial cells can be, for example, alpha-helical linear peptides (e.g., LL-37 or Ceropin P1), peptides containing disulfide bonds (e.g., alpha-defensins, beta-defensins or bacteriocins) Or peptides containing only one or two major amino acids (for example, PR-39 or indocyanin). Cell-penetrating peptides may also include a nuclear localization signal (NLS). For example, cell-penetrating peptides that are both amphipathic, such as MPG, are based on fusion peptide domains derived from the NLS of HIV-1 gp41 and SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31 :2717-2724, 2003).

C. Carbohydrate conjugates

In some embodiments of the compositions and methods of the invention, the iRNA further comprises carbohydrates. As described herein, the iRNA-conjugated carbohydrate facilitates in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that itself consists of one or more carbon atoms (which may be linear, branched, or cyclic) and oxygen, nitrogen, or sulfur atoms with each Carbohydrates composed of monosaccharide units bonded by carbon atoms Compounds; or compounds containing as part of a carbohydrate consisting of one or more monosaccharide units (which may be linear, branched or cyclic) containing at least six carbon atoms, each of which has an oxygen attached to it. , nitrogen or sulfur atoms. Representative carbohydrates include carbohydrates (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing approximately 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, and cellulose and polysaccharide gum. Specific monosaccharides include C5 and above (eg, C5, C6, C7, or C8) sugars; disaccharides and trisaccharides include sugars containing two or three monosaccharide units (eg, C5, C6, C7, or C8).

In some embodiments, the carbohydrate conjugates used in the compositions and methods of the present invention are monosaccharides.

In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). The GalNAc conjugate, which contains one or more N-acetyl galactosamine (GalNAc) derivatives, is described, for example, in US Pat. No. 8,106,022, the entire content of which is hereby incorporated by reference. In some embodiments, the GalNAc conjugate serves as a ligand for targeting iRNA to specific cells. In some embodiments, the GalNAc conjugate targets the iRNA to hepatocytes, e.g., as a ligand for the asialoglycoprotein receptor of liver cells (eg, hepatocytes).

In some embodiments, the carbohydrate conjugate includes one or more GalNAc derivatives. The GalNAc derivative can be connected via a linker, for example, a divalent or trivalent branched chain linker. In some embodiments, the GalNAc conjugate is attached to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is coupled to the iRNA agent (e.g., at the 3' end of the sense strand) via a linker, e.g., a linker as described herein. In some embodiments, the GalNAc conjugate is attached to the 5' end of the sense strand. In some implementations, the GalNAc The conjugate is attached to the iRNA agent (e.g., at the 5' end of the sense strand) via a linker, e.g., a linker as described herein.

In some embodiments of the present invention, the GalNAc or GalNAc derivative is connected to the iRNA agent of the present invention through a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is connected to the iRNA agent of the invention through a bivalent linker. In other embodiments of the present invention, the GalNAc or GalNAc derivative is connected to the iRNA agent of the present disclosure through a trivalent linker. In another embodiment of the present disclosure, the GalNAc or GalNAc derivative is connected to the iRNA agent of the present invention through a tetravalent linker.

In some embodiments, double-stranded RNAi agents of the invention include a GalNAc or GalNAc derivative linked to the iRNA agent. In some embodiments, the double-stranded RNAi agent of the present disclosure includes multiple (eg, 2, 3, 4, 5 or 6) GalNAc or GalNAc derivatives, each independently connected to the double-stranded RNAi by a plurality of monovalent linkers. multiple nucleotides of the agent.

In some embodiments, for example, when the two strands of the iRNA agent of the invention are connected to each other by forming a hairpin loop of nucleotides between the 3'-end of one strand and the corresponding 5'-end of the other strand A portion of an uninterrupted chain of a larger molecule containing multiple unpaired nucleotides. Each unpaired nucleotide within the hairpin loop may independently include GalNAc or a GalNAc derivative linked by a monovalent linker. . The hairpin loop may also be formed by an expanded overhang of one strand of the duplex.

In some embodiments, for example, when the two strands of the iRNA agent of the invention are connected to each other by forming a hairpin loop of nucleotides between the 3'-end of one strand and the corresponding 5'-end of the other strand A portion of an uninterrupted chain of a larger molecule containing multiple unpaired nucleotides. Each unpaired nucleotide within the hairpin loop may independently include GalNAc or a GalNAc derivative linked by a monovalent linker. . The hairpin loop may also be formed by an expanded overhang of one strand of the duplex.

In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is selected from the group consisting of:

，其中Y係O或S且n係3至6(式XXIV)；

, where Y is O or S and n is 3 to 6 (Formula XXIV);

，其中Y係O或S且n係3至6(式XXV)；

, where Y is O or S and n is 3 to 6 (Formula XXV);

，其中X係O或S(式XXVII)；

, where X is O or S (Formula XXVII);

In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is N-acetylgalactosamine, such as

In some embodiments, the RNAi agent is linked to the carbohydrate conjugate via a linker as shown below, where X is O or S.

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and is represented by:

Another representative carbohydrate conjugate for use in embodiments described herein includes, but is not limited to,

(Formula XXXVI), when one of X or Y is an oligonucleotide, the other is hydrogen.

In some embodiments, suitable ligands are those disclosed in WO2019/055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand includes the following structure:

In some embodiments of the present invention, the GalNAc or GalNAc derivative is connected to the iRNA agent of the present disclosure through a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is connected to the iRNA agent of the present disclosure via a bivalent linker. In other embodiments of the disclosure, the GalNAc or GalNAc derivative is linked to the iRNA agent of the disclosure via a trivalent linker.

In one embodiment, double-stranded RNAi agents of the invention comprise one or more GalNAc or GalNAc derivatives linked to an iRNA agent. The GalNAc can be connected to the nucleotides of the sense or antisense strand through a linker. This GalNAc can be connected to the 5'-end of the sense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, or the 3'-end of the antisense strand. In one embodiment, the GalNAc is linked to the 5'-end of the sense strand, for example, via a trivalent linker.

In other embodiments, the double-stranded RNAi agents of the invention comprise multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently linked by multiple streets to the double-stranded RNAi agent. of multiple nucleotides, for example, via monovalent linkers.

In some embodiments, for example, when the duplex of the iRNA agent of the present disclosure is part of a molecule, the molecule is linked at a 3'-end and a corresponding 5'-end by an uninterrupted nucleotide chain. Between the end and the other strand, a hairpin loop is formed, including multiple unpaired nucleosides Each unpaired nucleotide in the hairpin loop may independently include a GalNAc or GalNAc derivative linked via a monovalent linker.

In some embodiments, the carbohydrate conjugate complex contains one or more additional ligands as described above, such as, but not limited to, PK modulators or cell-penetrating peptides.

Additional carbohydrate conjugates and linkers suitable for use in the present disclosure include those described in PCT Publication Nos. WO2014/179620 and WO2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linker

In some embodiments, a conjugate or ligand as described herein is linked to an iRNA oligonucleotide with multiple cleavable or non-cleavable linkers.

The term "linker" or "linking group" means an organic moiety that links two parts of a compound together, eg, covalently attaches two parts of a compound. Linkers typically include direct bonds or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, _SO2 , _SO2NH , or chains of atoms such as, but not limited to, substituted or Unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroaryl alkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylaryl Alkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, Alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkyl Heterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocycle Alkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, one or more methylene groups can be composed of O, S, S(O), SO ₂ , N(R8), C(O), substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted heterocyclyl group continues or terminates; wherein R8 is hydrogen, hydroxyl, aliphatic or substituted aliphatic. In some aspects, the linker is about 1 to 24 atoms, 2 to 24 atoms, 3 to 24 atoms, 4 to 24 atoms, 5 to 24 atoms, 6 to 24 atoms, 6 to 18 atoms, 7 to 18 atoms, 8 to 18 atoms, 7 to 17 atoms, 8 to 17 atoms, 6 to 16 atoms, 7 to 17 atoms, or 8 to 16 atoms.

The cleavable linker is stable enough outside the cell, but upon entering the target cell it is cleaved to release the two parts held together by the linker. In a preferred aspect, the cleavage ratio of the cleavable linking group in the target cell or under first reference conditions (which may, for example, be selected to simulate or represent conditions within the cell) is greater than the cleavage ratio in the subject's blood. cleavage is at least about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times faster within or under second reference conditions (which may be, for example, selected to simulate or represent conditions found in blood or serum). 70 times, 80 times, 90 times or higher, or at least about 100 times.

Cleavable linking groups are those that are sensitive to cleavage agents such as pH, redox potential, or the presence of degradable molecules. Typically, lytic agents are more prevalent or found at higher magnitudes or activities within cells than in serum or blood. Examples of such degrading agents include redox agents that are selected for a particular substrate or that are not substrate specific, including, for example, degradable redox agents present within cells that can be cleavable by reduction of cleavable linker groups. Groups of oxidases, reductases or reducing agents such as thiols; esterases; endonucleases, or agents that create an acidic environment such as resulting in a pH of 5 or more Lower ones; enzymes that hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate specific), and phosphatases.

Cleavable linking groups such as disulfide bonds may be pH sensitive. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from 7.1 to 7.3. Endosomes have a more acidic pH, in the range of 5.5 to 6.0; lysosomes have an even more acidic pH, around 5.0. Some linkers will have cleavable linking groups that cleave at a preferred pH, thereby releasing the cationic lipid from the ligand within the cell or releasing the cationic lipid into the desired cell. inside the chamber.

Linkers may include cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into the linker may depend on the cell to be targeted. For example, the liver-targeting ligand can be linked to the linker via a linker that includes an ester group. Hepatocytes are rich in esterases, so linkers will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

When the target cell type is peptidase-rich, such as hepatocytes and synovial cells, linkers containing peptide bonds can be used.

Generally, the suitability of an alternative cleavable linking group can be assessed by testing the ability of a degrading agent (or condition) to cleave an alternative linking group. It is also desirable to test the ability of the alternative cleavable linkage group to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative sensitivity of lysis can be determined between a first condition selected as a lysis marker within a target cell and a second condition selected as another tissue or biological fluid such as blood. or cleavage markers in serum. Such assessments can be performed in cell-free systems, in cells, in cell cultures, in organ or tissue cultures, or in whole animals. Can To be useful, initial assessments can be made under cell-free or culture conditions and confirmed by further assessments in whole animals. In preferred embodiments, candidate compounds may have a greater cleavage ratio within cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions). (Bottom) is at least about 2x, 4x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or about 100x faster.

i. Redox-cleavable linking group

In some embodiments, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a linking group that can be cleaved by reduction is a disulfide linking group (-S-S-). To determine whether an alternative cleavable linking group is a suitable "reductively cleavable linking group" or, for example, suitable for use with a specific iRNA moiety and a specific targeting agent, the methods disclosed herein can be reviewed. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which incubation simulates the lysis that would be observed within cells, such as target cells. rate. The candidates can also be evaluated under conditions selected to simulate blood or serum conditions. In one embodiment, the candidate compound is cleaved in blood by up to about 10%. In other embodiments, candidate compounds may be degraded within cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions). ) is at least about 2x, 4x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or about 100x faster. The cleavage rate of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to simulate intracellular media and compared to that measured under conditions selected to simulate extracellular media.

ii. Cleavable linking groups based on phosphate esters

In certain embodiments, the cleavable linker comprises a phosphate-based cleavable linker group. Cleavable linkage groups based on phosphate are cleaved by agents that degrade or hydrolyze the phosphate group. Examples of agents that cleave phosphate groups within cells are enzymes such as intracellular phosphatases. Examples of phosphate linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O )(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)( ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk) -O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-. The preferred embodiment is -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH) -O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O -, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. Such candidates may be evaluated using methods similar to those described above.

iii. Acid-cleavable linking groups

In some embodiments, the cleavable linker includes an acid-cleavable linkage group. Acid-cleavable linking groups are linking groups that are cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or lower (eg, about 6.0, 5.75, 5.5, 5.25, 5.0 or lower), or Reagents such as enzymes can be used as universal acids for cleavage. Within cells, specialized low-pH organelles such as endosomes and lysosomes provide an environment for the cleavage of acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid-cleavable linking group may have the general formula -C=NN-, C(O)O, or -OC(O). Preferred practice is when the carbon-based aryl group attached to the ester oxygen (alkoxy) Aspects are substituted alkyl, or quaternary alkyl such as dimethylpentyl or tertiary butyl. Such candidates may be evaluated using methods similar to those described above.

iv. Cleavable linking groups based on esters

In certain embodiments, the cleavable linker comprises an ester-based cleavable linker group. Ester-based cleavable linkage groups are cleaved intracellularly by enzymes such as esterases or amidases. Examples of ester-based cleavable linking groups include, but are not limited to, alkylene, alkenylene, and alkynylene esters. The ester-cleavable linking group may have the general formula -C(O)O- or -OC(O)-. Such candidates may be evaluated using methods similar to those described above.

v. Cleavable linking groups based on peptides

In yet another embodiment, the cleavable linker includes a peptide-based cleavable linker group. Peptide-based cleavable linking groups are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable groups are formed between amino acids to obtain peptide bonds for oligopeptides (eg, dipeptides, tripeptides, etc.) and polypeptides. Cleavable groups based on peptides do not include amide groups (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene groups. Peptide bonds are formed between amino acids to obtain specific types of amide bonds in peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (ie, amide bonds) formed between amino acids to obtain peptides and proteins, and do not include the intact amide functionality. The cleavable linking group based on the peptide has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. Such candidates may be evaluated using methods similar to those described above.

In yet another embodiment, the iRNA of the invention is conjugated to the carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to,

(式XLI)、

(Formula XLI),

(Formula XLIV), when one of X or Y is an oligonucleotide and the other is hydrogen.

In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) linked via a divalent or trivalent branched chain linker. derivative.

In one embodiment, the dsRNA of the present invention is coupled to a linker of a bivalent or trivalent branch chain selected from the group consisting of the structures shown in any one of formulas (XLV) to (XLVI):

in:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent 0 to 20 each time they appear, where the repeating units can be the same or different;

P ^2A , P ^2B , P3A , P ^3B , P ^4A , P ^4B , P ^5A , P ^5B , P ^5C , T 2A , T ^2B , T ^3A , T ^3B , T ^4A , T ^4B , T ^4A , ^{T 5B ,} T ^5C is independently absent, CO, NH, O, S, OC(O), NHC(O), CH ₂ , CH ₂ NH or CH ₂ O on each occurrence;

Q ^2A , Q ^2B , Q ^3A , Q ^3B , Q ^4A , Q ^4B , Q ^5A , Q ^5B , Q ^5C are independently absent or alkylene or substituted alkylene (where, One or more methylene groups can be represented by one of O, S, S(O), SO ₂ , N(RN), C(R')=C(R”), C≡C or C(O) or more interruption or termination;

、

、

、

、

或雜環基； R ^2A , R ^2B , R ^3A , R ^3B , R ^4A , R ^4B , R ^5A , R ^5B , R ^5C are independently absent each time they appear, NH, O, S, CH ₂ , C(O)O , C(O)NH, NHCH(R ^a )C(O), -C(O)-CH(R ^a )- NH-, CO, CH=NO,

,

,

,

,

or heterocyclyl;

L ^2A , L ^2B , L ^3A , L ^3B , L ^4A , L ^4B , L ^5A , L ^5B and L ^5C represent ligands, that is, each time they appear, they are independently monosaccharides (such as GalNAc), diose Sugar, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R ^a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives can especially be used in combination with RNAi agents to inhibit the expression of target genes, such as those of formula (XLIX):

Where L ^5A , L ^5B and L ^5C represent monosaccharides, such as GalNAc derivatives.

Examples of suitable linkers joining the divalent and trivalent branched chains of GalNAc derivatives include, but are not limited to, the structures cited above as Formulas II, VII, XI, X, and XIII.

Representative U.S. patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, No. 5,580,731, No. 5,591,584, No. 5,109,124, No. 5,118,802, No. 5,138,045, No. 5,414,077, No. 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,5 No. 87,044, No. 4,605,735, No. 4,667,025 No. 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013, 5,082,830, 5,112,963, 5,214,136, 5 , No. 082,830, No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,5 No. 12,667, No. 5,514,785, No. 5,565,552 , No. 5,567,810, No. 5,574,142, No. 5,585,481, No. 5,587,371, No. 5,595,726, No. 5,597,696, No. 5,599,923, No. 5,599,928, No. 5,688,941, No. 6,294,664, No. 6 , No. 320,017, No. 6,576,752, No. No. 6,783,931, No. 6,900,297, No. 7,037,646, and No. 8,106,022, the entire contents of each of which are incorporated herein by reference.

It is not necessary that all positions of a given compound be modified uniformly, and in fact, more than one of the foregoing modifications may be incorporated into a single compound or even at a single nucleoside of the iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

In the context of this disclosure, "chimeric" iRNA compounds or "chimeras" are iRNA compounds, preferably dsRNA agents, that contain two or more chemically distinct regions, Each is composed of at least one monomer unit, that is, in the case of dsRNA compounds, this monomer unit is a nucleotide. Such iRNAs typically contain at least one region in which the RNA is modified to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased affinity for the target nucleic acid. Additional regions of iRNA can serve as substrates for enzymes capable of cleaving RNA:DNA hybrids or RNA:RNA hybrids. For example, RNaseH is a cellular endonuclease that cleaves the RNA strand of the RNA:DNA double helix. Therefore, activation of RNaseH results in cleavage of the RNA target, thereby greatly increasing the efficiency of iRNA inhibition of gene expression. Therefore, when using chimeric dsRNA, using shorter iRNAs often results in comparable results to using phosphorothioate deoxydsRNA that hybridizes to the same target region. Cleavage of the RNA target can be generally checked by gel electrophoresis, and if necessary, gel electrophoresis can be combined with relevant nucleic acid hybridization techniques known in the art.

In some cases, the RNA of iRNA can be modified by non-ligand groups. A large number of non-ligand molecules have been conjugated to iRNA to enhance iRNA activity, cellular distribution, or cellular uptake, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1): 54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86: 6553), cholic acid (Manoharanetal., Bioorg. Med. Chem. Lett., 1994, 4: 1053), thioethers such as hexyl-S-tritylmercaptan (Manoharanetal., Ann.N.Y.Acad.Sci., 1992, 660: 306; Manoharanetal., Bioorg.Med.Chem.Let., 1993, 3: 2765), thiocholesterol (Oberhauseretal., Nucl.AcidsRes., 1992 , 20: 533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBOJ., 1991, 10: 111; Kabanov et al., FEBS Lett., 1990, 259 : 327; Svinarchuk et al., Biochimie, 1993, 75: 49), phospholipids such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3- Triethylammonium phosphate (Manoharanetal., Tetrahedron Lett., 1995, 36: 3651; Sheaetal., Nucl. Acids Res., 1990, 18: 3777), polyamine or polyethylene glycol chains (Manoharanetal., Nucleosides & Nucleotides, 1995, 14: 969), or adamantane acetic acid (Manoharanetal., Tetrahedron Lett., 1995, 36: 3651), palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229), or 18 Alkylamine or hexylamine-carbonyloxycholesterol moiety (Crookee et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative US patents teaching the preparation of such RNA conjugates are listed above. Typical engagement strategies involve one or more of the sequences Synthesis of RNA carrying an amine linker at each position. The amine group then reacts with the molecule conjugated using a suitable coupling agent or activator. The ligation reaction can be performed with the RNA still bound to the solid support, or after the RNA has been cleaved in the solution phase. Purification of RNA conjugates by HPLC typically provides pure conjugates.

IV. Delivery of RNAi Agents of the Disclosure

Delivery of RNAi agents of the invention to cells, such as cells within an individual, such as a human subject (e.g., an individual in need thereof), such as an individual susceptible to or diagnosed with an AGT-related disorder, e.g., hypertension, can be achieved by There are many different ways to do this. For example, delivery can be performed by contacting cells with the iRNA of the present disclosure in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition comprising an RNAi group, such as dsRNA, to an individual. Alternatively, in vivo delivery can be performed indirectly by administration of one or more vectors that encode and direct the expression of the RNAi agent. These options are reviewed further below.

In general, any method of delivering nucleic acid molecules (in vitro or in vivo) is suitable for use with the RNAi agents of the present disclosure (see, e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139 -144 and WO94/02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors considered for delivery of an RNAi agent include, for example, biological stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent within the target tissue. RNA interference has also shown success in local delivery by guided injection into the CNS (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59 -66; Makimura, H. et a.l (2002) BMC Neurosci. 3: 18; Shishkina, GT., et al. (2004) Neuroscience 129: 521-528; Thakker, ER., et al. (2004) Proc. Natl.Acad.Sci.U.S.A.101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93: 594-602). Modifications of RNA or drug carriers may also allow RNAi agents to target tissues and avoid undesirable off-target effects. RNAi molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, systemic injection of an anti-ApoB RNAi agent conjugated to a lipophilic cholesterol moiety into mice resulted in attenuation of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432 :173-178).

In another embodiment, the iRNA agent can be delivered using a drug delivery system such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. The positively charged cation delivery system promotes the binding of iRNA molecules (negatively charged) and also enhances interactions at the negatively charged cell membrane to allow efficient uptake of iRNA by the cell. Cationic lipids, dendrimers or polymers can be bound to iRNA or induced to form vesicles or vesicles that enclose iRNA (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2) :107-116). The formation of vesicles or micelle further prevents degradation of the RNAi agent when administered systemically. Methods of making and administering cationic-RNAi agent complexes are well within the capabilities of those of ordinary skill in the art (see, e.g., Sorensen, DR., et al. (2003) J. Mol. Biol 327:761 -766; Verma, UN. et al., (2003) Clin. Cancer Res. 9: 1291-1300; Arnold, AS et al. (2007) J. Hypertens. 25: 197-205, all by reference incorporated herein in its entirety). Some non-limiting examples of drug delivery systems that can be used for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003), supra), " Solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441: 111-114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther. 12: 321-328; Pal , A.et al., (2005) Int J.Oncol.26: 1087-1091), polyethylenimine (Bonnet ME.et al., (2008) Pharm.Res.Aug 16 electronic priority release; Aigner, A . (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3: 472-487) and polyamide-based amine (Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35: 61-67; Yoo, H. et al., (1999) Pharm. Res. 16: 1799-1804). In some embodiments, iRNAi forms a complex with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety. Certain aspects of the disclosure relate to methods of reducing AGT gene expression in a cell, comprising contacting the cell with a double-stranded RNAi agent of the disclosure. In one embodiment, the cell is a cell of the liver, optionally a hepatocyte. In one embodiment, the cells are extrahepatic cells.

A. Vectors encoding iRNAs of the present invention

iRNA targeting the AGT gene can be expressed from a transcription unit inserted into a DNA or RNA vector (see, for example, Couture, A, et al., TIG. (1996), 12: 5-10; Skillern, A, et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, US Patent No. 6,054,299). Better performance may be transient (hours to weeks) or sustained (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These gene transfections can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating vectors or non-integrating vectors. Transgenes can also be constructed to allow their inheritance as mitochondrial exoplasts (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, etc.; (c) Adeno-associated virus vector; (d) Herpes simplex virus vector; (e) SV40 vector; (f) Polyoma virus vector; (g) Papilloma virus vector; (h) Picornavirus vector; ( i) poxvirus vectors, such as smallpox, such as vaccinia virus vectors, or fowlpox, such as canarypox or fowlpox virus vectors; and (j) helper-dependent or naked adenovirus vectors. Replication-deficient viruses can also be dominant. Different vectors will or will not become incorporated into the genome of the cell. If necessary, these constructs may include viral sequences for transfection. Alternatively, the construct can be incorporated into vectors capable of episomal replication, such as EPV vectors and EBV vectors. Constructs for recombinant expression of RNAi agents will typically require regulatory elements such as promoters, enhancers, etc., to ensure expression of the RNAi agent within the target cell. Other considerations for carriers and construction are known in the art.

V. Pharmaceutical composition of the present invention

The invention also includes pharmaceutical compositions and preparations including the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing iRNA as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing the iRNA can be used to prevent or treat AGT-related disorders, such as hypertension.

The pharmaceutical composition is formulated based on the delivery mode. One embodiment is to formulate the composition for systemic administration via parenteral delivery, for example, by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical composition of the present invention can be administered at a dose sufficient to inhibit the expression of the AGT gene.

In some embodiments, the pharmaceutical compositions of the present invention are sterile. In another embodiment, the pharmaceutical compositions of the present invention are pyrogen-free.

The pharmaceutical composition of the present invention can be administered at a dose sufficient to inhibit the expression of the AGT gene. Generally, a suitable dose of the iRNA of the invention will be in the range of about 0.001 to about 200.0 mg per kilogram of the recipient's body weight per day, typically in the range of about 1 to 50 mg per kilogram of the recipient's body weight per day. Generally speaking, suitable dosages of iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, such as about 0.3 mg/kg and about 3.0 mg/kg. Repeat dosing regimens may include administration of therapeutic amounts of iRNA at regular intervals, such as monthly, every 3 to 6 months, or annually. In some embodiments, the iRNA is administered from about once a month to about every six months.

After the initial treatment regimen, the treatment may be administered less frequently. The duration of treatment can be determined based on the severity of the disease.

In other embodiments, a single dose of the pharmaceutical composition may be administered over an extended period of time such that the interval between administrations does not exceed 1, 2, 3, or 4 months. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a month. In other embodiments of the invention, single doses of the pharmaceutical compositions of the invention are administered quarterly (ie, about every three months). In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered twice per year (i.e., approximately once every six months).

Those with ordinary knowledge should be aware that certain factors may affect the dosage and timing required to effectively treat an individual, including but not limited to the presence of mutations in the individual, previous treatments, the general health or age of the individual, and the presence of other diseases. Furthermore, treating an individual with a prophylactically or therapeutically effective amount of a composition may involve a single treatment or a series of treatments.

The pharmaceutical compositions of the present disclosure may be administered via a variety of routes, depending on whether local or systemic treatment is desired, and depending on the area to be treated. The drug can be administered externally (including Ophthalmic, vaginal, rectal, intranasal, transdermal); oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, for example, by implanted devices; or intracranial, for example, by intraparenchymal, intrathecal or intraventricular administration. .

The iRNA can be delivered in a manner that targets specific tissues, such as the liver.

Pharmaceutical compositions and preparations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Traditional medical carriers, aqueous bases, powder bases, oily bases, thickeners, etc. may be necessary or desired. Coated condoms, gloves, etc. can also be used. Suitable topical formulations include those in which the RNAi agents proposed by the present disclosure are mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleyl phospholipid DOPE ethanolamine, dimyristyl lecithin DMPC, distearyl lecithin), negative (e.g., dimyristyl phospholipid glycerol DMPG ) and cationic (for example, dioleyltetramethylaminopropyl DOTAP and dioleylphospholipid ethanolamine DOTMA). The RNAi agents proposed in the present disclosure can be encapsulated in liposomes or can form complexes with liposomes, especially cationic liposomes. Alternatively, the RNAi agent can be complexed with lipids, especially cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, arachidic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, perlenic acid, di- Capric acid ester, tricapric acid ester, glyceryl monooleate, glyceryl dilaurate, glyceryl 1-monocanoate, 1-dodecyl azepan-2-one, acylcarnitine, acylcarnitine Choline, or its C1-20 alkyl ester (for example, isopropyl myristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt. Topical formulations are disclosed in detail in US 6,747,014, which is incorporated herein by reference.

In one embodiment, the siRNAs (double-stranded RNA agent of the present invention) are administered into cells via a local administration route as a pharmaceutical composition.

In one embodiment, the pharmaceutical composition may include a siRNA compound mixed with a topical delivery agent. The external delivery agent can be composed of multiple microscopic vesicles. The microscopic vesicles can be liposomes. In some embodiments, the lipid system is a cationic liposome.

In additional embodiments, the dsRNA agent is mixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, didecanoate, trioctanoate, monooil Acid, glyceryl dilaurate, 1-glyceryl monodecanoate, 1-dodecylazepan-2-one, acylcarnitine, acylcholine or C1 to 10 alkyl ester, glycerin Monoesters, diglycerides or pharmaceutically acceptable salts thereof.

In another embodiment, the topical penetration enhancer is bile salts. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, gluconic acid, glycolic acid, glycolic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, urside Oxycholic acid, sodium taurine-24,25-dihydrofucinate, sodium dihydrofucinate, polyoxyethylene-9-lauryl ether or pharmaceutically acceptable salts thereof.

In other embodiments, the topical penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, salicylate, N-acyl derivatives of collagen, laureth-9, N-amino acyl derivatives of β-diketone, or mixtures thereof.

In other embodiments, the penetration enhancer is a surfactant, such as an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, perfluoride emulsion or a mixture thereof.

In other embodiments, the penetration enhancer can be selected from the group consisting of unsaturated cyclic ureas, 1-alkyl-alkanones, 1-alkenyl azacyclo-propanone, steroid anti-inflammatory agents, and mixtures thereof group. In yet another embodiment, the penetration enhancer can be ethylene glycol, pyrrole, azone or terpene.

In one aspect, the invention features a pharmaceutical composition (e.g., a precursor, e.g., a larger siRNA compound that can be processed into an ssiRNA compound, or an encoding DNA of siRNA compounds, for example, double-stranded siRNA compounds, or ssiRNA compounds, or precursors thereof). In one embodiment, the injectable dosage form of the pharmaceutical composition includes a sterile aqueous solution or dispersion and a sterile powder. In some embodiments, the sterile aqueous solution includes a diluent such as water; physiological saline, fixed oil, polyethylene glycol, glycerin, or propylene glycol.

The iRNA molecules of the invention can be incorporated into pharmaceutical compositions. This composition typically includes one or more iRNAs and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with the pharmaceutical administration to cells. Contains, for example, liver cells. Such media and agents for use in pharmaceutical active substances are well known in the art. Unless any general media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing preparations. These compositions can be produced from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. Formulations include those targeting the liver.

The pharmaceutical preparations of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to general techniques well known in the pharmaceutical industry. This technology involves the step of combining the active ingredient with a pharmaceutical carrier or excipient. Generally, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier.

A. Other preparations

i.Lotion

The compositions of the present invention can be prepared and formulated as emulsions. An emulsion is a typical heterogeneous system in which one liquid is dispersed in another liquid in the form of droplets typically exceeding 0.1 μm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC.,2004,Lippincott Williams & Wilkins(8th ed.),New York,NY;Idson,Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker(Eds.),1988,Marcel Dekker,Inc.,New York,N.Y.,volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman ,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p.335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985 , p.301). Emulsions are generally biphasic systems containing two immiscible liquid phases that are intimately mixed and dispersed with each other. Typically, emulsions can be of the water-in-oil (w/o) type or oil-in-water (o/w) type. When the aqueous phase is finely divided into small droplets and dispersed throughout the oil phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when the oily phase is finely divided into small droplets and dispersed throughout the aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. In addition to containing dispersed phases, emulsions can be used as aqueous phases, oils The active phase may also contain additional components in addition to the active drug present in solution or as a separate phase by itself. If necessary, pharmaceutical excipients such as emulsifiers, stabilizers, dyes and antioxidants can also be present in the emulsion. Pharmaceutical emulsions can also be multi-emulsions composed of more than two phases, for example, water-in-oil (o/w/o) emulsions and water-in-oil-in-water (w/o/w) emulsions. Such formulations often provide certain advantages that simple biphasic emulsions do not have. Multiple emulsions in which individual oil droplets of o/w emulsion accommodate small water droplets constitute w/o/w emulsions. Likewise, oil droplets are contained within a system of water globules that are stabilized within the oily continuous phase, providing an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Generally, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase and maintains its form by means of emulsifiers or by means of viscosity formation. Either phase of the emulsion may be semi-solid or solid, as in the case of lotion-type ointment bases and creams. Other means of stabilizing emulsions require the use of emulsifiers that can be incorporated into either phase of the emulsion. Broadly speaking, emulsifiers can be classified into four categories: synthetic surfactants, natural surfactants, absorbent matrices, and finely divided solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG ., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surfactants, have been widely used in emulsion formulations and have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York ,N.Y.,1988,volume 1,p.199). Surfactants are typically amphiphilic and contain a hydrophilic part and a hydrophobic part. The ratio of hydrophilicity to hydrophobicity of a surfactant has been defined as the hydrophilic/lipophilic balance (HLB) and is a valuable tool in classifying and selecting surfactants in the preparation of formulations. Based on the nature of their hydrophilic groups, surfactants can be classified into different categories: nonionic, anionic, cationic, and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

A number of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

The use of emulsion formulations via skincare, oral and parenteral routes and methods of their manufacture have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC. ,2004,Lippincott Williams & Wilkins(8th ed.),New York,NY;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

ii. Microemulsion

In one embodiment of the invention, the composition of iRNAs and nucleic acids is formulated as a microemulsion. Microemulsions can be defined as aqueous, oily, and amphoteric systems that are single optically isotropic and thermodynamically stable solutions (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC .,2004,Lippincott Williams & Wilkins(8th ed.),New York,NY;Rosoff,in Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker(Eds.),1988,Marcel Dekker,Inc.,New York,N.Y.,volume 1, p.245). Typically, microemulsions are systems prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth ingredient, usually a medium chain length alcohol, to form a transparent system. Microemulsions have therefore also been revealed to be thermodynamically stable, isotropic, clear dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, Controlled Release of Drugs : Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).

iii.Particles

The iRNA of the invention can be incorporated into particles, for example, microparticles. Microparticles can be produced by spray drying, but can also be produced by other methods including freeze-drying, evaporation, fluid bed drying, vacuum drying or combinations of these techniques.

iv. Penetration enhancer

In one embodiment, the present invention uses a variety of penetration enhancers to achieve effective delivery of nucleic acids, especially iRNAs, to animal skin. Most drugs are classified as ionized or non-ionized Two forms exist in solution. However, generally only fat-soluble or lipophilic drugs easily cross cell membranes. It has been found that even non-lipophilic drugs can still cross the cell membrane if the cell membrane to be crossed is treated with a permeation enhancer. In addition to facilitating the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the penetration ability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories: namely, surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above categories of penetration enhancers and their use in the manufacture of pharmaceutical compositions and delivery of pharmaceutical agents are well known in the art.

vi. Excipients

In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or other pharmaceutically inert vehicle used to deliver one or more nucleic acids to an animal. things. The excipient may be liquid or solid and is selected to provide the desired volume, consistency, etc., when combined with the nucleic acid and other components of a given pharmaceutical composition, based on the intended mode of administration contemplated. Such agents are well known in the technical field.

vii.Other components

The compositions of the present invention may additionally contain other auxiliary components commonly found in pharmaceutical compositions at concentrations commonly used in this field. Thus, for example, such compositions may contain additional, compatible, pharmaceutically active materials such as, for example, antipruritic, astringent, local anesthetic, or anti-inflammatory agents, or may contain materials useful in physical formulations. Various types of compositions of the present invention Additional materials such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners and stabilizers. However, when such materials are added, such materials should not unduly interfere with the biological activity of the components of the compositions of the invention. The preparation can be sterilized and, if necessary, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts used to influence the osmotic pressure that do not adversely react with the nucleic acids of the preparation. , buffers, colorants, flavoring agents or aromatic substances.

Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension may also contain stabilizers.

In some embodiments, the pharmaceutical compositions provided by the present invention include (a) one or more iRNAi and (b) one or more agents that function through non-iRNA mechanisms and are useful for treating AGT-related disorders. , for example, hypertension.

The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, determining the LD50 (the dose that is lethal to 50% of a population) and the ED50 (the dose that is therapeutically effective for 50% of a population). . The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, and it can be expressed as the ratio LD50/ED50. Compounds that exhibit a high therapeutic index are preferred.

Data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for use in humans. The dose of the composition proposed by the present invention is usually within a circulating concentration range of low or no toxicity including ED50, such as ED80 or ED90. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound used in the methods proposed by this invention, the therapeutically effective dose can be constructed initially from cell culture assays. Doses can be formulated in animal models to achieve a range of circulating plasma concentrations of the compound or, when appropriate, the polypeptide product of the target sequence (e.g., to achieve a reduced concentration of the polypeptide), which range includes Included are the IC50 (ie, the concentration of the test compound at which half-maximal inhibition of a symptom is achieved) as determined in cell culture or a higher magnitude of inhibition as determined in cell culture. Such information can be used to more accurately determine doses that can be administered to humans. For example, levels in plasma can be measured by high performance liquid chromatography.

In addition to administration as discussed above, the iRNA proposed by the present invention can also be administered in combination with other known preventive methods or treatments for AGT-related disorders. In any event, the attending physician can determine the amount and timing of iRNA administration based on observations using standard efficacy measurement methods known in the art or described herein.

VI. Methods to inhibit the expression of AGT

The present invention also provides methods for inhibiting the expression of AGT genes in cells. The method includes contacting a cell with an RNAi agent (eg, a double-stranded RNAi agent) in an amount sufficient to inhibit the expression of AGT in the cell, thereby inhibiting the expression of AGT in the cell. In some embodiments of the present disclosure, AGT gene expression in the liver (eg, hepatocytes) is preferentially inhibited.

Contacting cells with iRNA, such as double-stranded RNAi agents, can occur in vitro or in vivo. Contacting cells with iRNA in vivo includes contacting cells or groups of cells within an individual, such as a human individual, with an RNAi agent. Combinations of methods of contacting cells in vitro and methods of contacting cells in vivo are also possible. Contact with cells can be direct or indirect, as discussed above. Furthermore, contact with cells can be via a targeting ligand, including any ligand disclosed herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, such as a GalNAc ligand, or any other ligand that directs the RNAi agent to the site of interest.

The term "inhibiting" as used herein is used interchangeably with "reducing," "silencing," "downregulating," "suppressing" and other similar terms. And includes suppression of any magnitude.

The phrase "inhibiting espressing of an AGT" means inhibiting the expression of any AGT gene (such as, for example, the following, mouse AGT gene, rat AGT gene, monkey AGT gene, or human AGT gene) as well as AGT The expression of gene variants or mutants. Therefore, the AGT gene may be a wild-type AGT gene, a mutant AGT gene, or a genetically modified AGT gene in the case of a genetically manipulated cell, cell population, or organism.

"Inhibiting expression of an AGT gene" includes any level of inhibition of the AGT gene, for example, at least partial inhibition of the expression of the AGT gene. Expression of the AGT gene can be assessed based on the magnitude or change in magnitude of any variable associated with AGT gene expression, such as AGT mRNA magnitude or AGT protein magnitude. This magnitude can be detected in a single cell or in a group of cells, including, for example, in a sample from an individual.

Suppression can be assessed by a reduction in one or more variables related to AGT performance compared to a control magnitude. The control level may be any type of control level used in the art, for example, a baseline level before dosing, or similarly measured in individuals, cells or samples treated with or without a control. Magnitude (e.g., buffer-only control or no-active control).

In some embodiments of the method of the invention, the expression of the AGT gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or Suppressed below the inspection level of the detection. In some embodiments, the expression of the AGT gene is inhibited At least 70%. It will also be understood that inhibition of AGT expression in some tissues (eg, liver) will not significantly inhibit expression in other tissues (eg, brain), where such inhibition is desired. In some embodiments, the detection method provided in Example 2 is used to determine the magnitude of performance in an appropriate species-matched cell line at a 10 nM siRNA concentration.

In some embodiments, in vivo performance is determined by human gene knockdown in rodents expressing the human gene, e.g., AAV-infected mice expressing the human target gene (i.e., AGT), e.g., When administered as a single dose. For example, give 3 mg/kg at the nadir of RNA performance. Knockdown of endogenous gene expression in model animal systems can also be determined, for example, by administering 3 mg/kg at the nadir of RNA expression after administration of a single dose. Such systems are useful when the nucleic acid sequences of the human gene and the model animal gene are sufficiently close such that the human iRNA provides efficient knockdown of the model animal gene.

Inhibition of AGT gene expression may be manifested by the amount of mRNA expressed by a first cell or population of cells (such cells may be present, for example, in a sample derived from an individual) in which the AGT gene is transcribed and has been processed (e.g., By contacting a cell or cell population with the iRNA of the invention, or by administering the iRNA of the invention to an individual in which cells exist or have existed), the expression of the AGT gene is inhibited, such as with a second cell or a third cell. Comparing a two-cell group, the second cell or second group of cells is substantially the same as the first cell or first group of cells but has not been or has not been so treated (the control cells have not been treated with iRNA or have not been targeted with interest. iRNA processing of genes). In some embodiments, the inhibition is assessed by the method provided in Example 2, in a species that matches the cell line and expresses the magnitude of the mRNA in the treated cells as a percentage of the magnitude of the mRNA in the control cells, using 10nM siRNA concentration and use the following formula:

In other embodiments, inhibition of AGT gene expression can be assessed functionally with a decrease in AGT gene expression, such as in HTT protein amounts and associated parameters in the blood and serum of an individual. AGT gene silencing can be measured in any cell expressing AGT endogenously or heterologously with the expression construct and by any assay known in the art.

Inhibition of AGT protein expression may be manifested by a reduction in the level of AGT protein expressed by a cell or population of cells (eg, the level of protein expressed in a sample derived from an individual). As noted above, for the assessment of mRNA repression, inhibition of protein expression magnitude in treated cells or cell populations can similarly be expressed as a percentage relative to protein expression magnitude in control cells or cell populations, e.g., as derived from its blood or serum.

Control cells or cell populations that can be used to assess inhibition of AGT gene expression include cells or cell populations that have not been contacted with the RNAi agent of the invention. For example, a control cell or population of cells may be derived from an individual individual (eg, a human or animal individual) prior to treatment of the individual with an RNAi agent or from an appropriately matched population control group.

The magnitude of AGT mRNA expressed by a cell or population of cells can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the magnitude of AGT expression in a sample is determined by detecting transcribed polynucleotides or portions thereof, such as the mRNA of the AGT gene. RNA can be extracted from cells using RNA extraction techniques including, for example, acid phenol/guanidinium isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kit (Qiagen®), or PAXgene ^™ (PreAnalytix ^™ , Switzerland). Typical detection formats using ribonucleic acid hybridization include nuclear conjugation detection, RT-PCR, RNase protection detection, Northern blot, in situ hybridization and microarray analysis.

In some embodiments, nucleic acid probes are used to determine the magnitude of AGT expression. As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific AGT nucleic acid or protein, or fragment thereof. Probes can be synthesized by one of ordinary skill in the art or derived from suitable biological agents. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used for hybridization or amplification assays including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for measuring mRNA levels involves contacting isolated mRNA with nucleic acid molecules (probes) that hybridize to HTT mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as a nitrocellulose membrane. In alternative embodiments, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, such as in an Affymetrix® chip array. One of ordinary skill can readily employ known mRNA detection methods to determine the magnitude of AGT mRNA.

Alternative methods of detecting the magnitude of AGT expression in a sample involve processes such as nucleic acid amplification or reverse transcriptase (to prepare cDNA) of the mRNA in the sample, e.g., by RT-PCR (in Mullis, 1987, US Patent No. 4,683,202 Experimental implementation described above), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193), autonomous sequence replication system (Guatelli et al. (1990) Proc. Natl. Acad Sci. USA 87: 1874-1878), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q- β replicase (Lizardi et al. ( 1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., US Patent No. 5,854,033) or any other nucleic acid amplification method, and then amplified using techniques commonly known to those of ordinary skill in the art. of molecules. These detection protocols are particularly useful for detecting nucleic acid molecules if these molecules are present in very low amounts. In a specific aspect of the invention, the level of AGT expression is detected by quantitative fluorescent RT-PCR (i.e., TaqMan™ System). In some embodiments, the magnitude of expression in species-matched cell lines is determined by the method provided in Example 2 using, for example, 10 nM siRNA.

The level of AGT mRNA expression can be monitored using membrane blots (such as those used in hybridization assays such as northern spots, southern spots, dots, etc.) or microwells, sample tubes, gels, beads or fibers (or Any solid support containing bound nucleic acids). See, U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which patents are incorporated herein by reference. Determination of AGT expression levels may also include nucleic acid probes in solution.

In some embodiments, branched DNA (bDNA) detection or real-time PCR (qPCR) is used to assess the magnitude of mRNA expression. The use of these methods is disclosed and exemplified in the Examples herein. In some embodiments, the magnitude of expression in species-matched cell lines is determined by the method provided in Example 2 using, for example, 10 nM siRNA.

The magnitude of AGT protein expression can be determined using any method known in the art for measuring protein magnitude. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, colorimetry Detection, spectrophotometry, flow cytometry, immunoexpansion Scattered (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence detection, electrochemiluminescence detection, etc.

In some embodiments, efficacy of the methods of the invention is measured by a decrease in AGT mRNA or protein levels (eg, in liver biopsies).

In some embodiments, efficacy of the methods of the invention can be monitored by detecting or monitoring a reduction in symptoms of AGT-related disorders. Monitoring the efficacy of a method by measuring any of these parameters or any combination of parameters is well within the capabilities of one of ordinary skill in the art.

In some embodiments of the methods of the invention, the iRNA is administered to an individual such that the iRNA is delivered to a specific site in each individual's body. Inhibition of AGT expression can be assessed using detection of, or changes in, magnitude of AGT mRNA or AGT protein in samples of fluid or tissue from a specific site in an individual (eg, liver or blood).

As used herein, the terms detecting or determining the magnitude of an analyte are understood to mean performing steps to determine the presence or absence of material, eg, protein, RNA. As used herein, checking or determining a method includes checking or determining an analyte level that is lower than the check level of the method used.

VII. Prevention and treatment methods of the present invention

The present invention also provides methods for inhibiting the expression of AGT using the iRNA of the present invention or compositions containing the iRNA of the present invention, thereby preventing or treating AGT-related disorders, such as hypertension. In the methods of the invention, cells can be contacted with siRNA in vitro or in vivo, that is, the cells can be within an individual.

Cells suitable for treatment using the methods of the invention can be any cell that expresses the AGT gene. Cells suitable for use in the methods of the present disclosure may be mammalian cells, such as primate cells (such as human cells or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (such as rat cells), rat cells or mouse cells). In some aspects, the cells are human cells, such as human liver cells. In the methods of the present invention, the expression of AGT in the cells is inhibited by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or to less than Check the magnitude.

In vivo methods of the present invention may include administering to an individual a composition containing an iRNA, wherein the iRNA includes a nucleotide sequence complementary to at least a portion of the RNA transcript of the AGT gene of the mammal to which the RNAi agent is to be administered. The composition may be administered by any means known in the art, including, but not limited to, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intracerebroventricular, intraparenchymal, and intrathecal), Intravenous, intramuscular, subcutaneous, transdermal, tracheal (aerosol), intranasal, intrarectal and topical (including buccal and sublingual) administration.

In some embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by subcutaneous injection. In some embodiments, the composition is administered by intramuscular injection.

The mode of administration can be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration can be selected to enhance targeting.

In one aspect, the invention also provides methods of inhibiting AGT gene expression in mammals. The method includes administering to a mammal a composition comprising a dsRNA targeting an AGT gene in a mammalian cell, and maintaining the mammal for a time sufficient to obtain degradation of the AGT gene's mRNA transcript, thereby inhibiting the AGT gene in the cell. Performance. Decreased gene expression can For example, qRT-PCR is assessed by any method known in the art and by the methods disclosed herein, eg, in Example 2. Reduction in protein product can be assessed by any method known in the art, such as ELISA. In some embodiments, a liver biopsy sample is used as tissue material to monitor a decrease in AGT gene or protein expression. In other embodiments, blood samples are used as individual samples to monitor reductions in AGT protein expression.

The present invention further provides methods of treatment for individuals in need thereof, for example, individuals diagnosed with AGT-related disorders, such as hypertension.

The present invention further provides methods of prevention in individuals in need thereof. The treatment methods of the present invention include administering the iRNA of the present invention to an individual, for example, administering a prophylactically effective amount of dsRNA targeting the AGT gene or a pharmaceutical composition comprising a dsRNA targeting the AGT gene to the individual, e.g., benefiting from AGT Individuals with reduced performance.

In one aspect, the present invention provides methods of treating individuals with disorders that would benefit from reduced performance of AGT, e.g., AGT-related disorders, hypertensive hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension Blood pressure, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, and hypoplasia Hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension related to renin activity or plasma renin concentration; hypertensive heart disease, hypertensive nephropathy, atherosclerosis sclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction , angina, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth retardation, obesity, Hepatic steatosis/fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome.

In some embodiments, the RNAi agent is administered to the subject in an amount effective to inhibit the expression of AGT in the subject. The amount effective to inhibit the expression of AGT in the cells of an individual can be assessed using the methods discussed above, including involving assessment of inhibition of AGT mRNA, AGT protein, or related variables, such as reducing the severity of symptoms of AGT-related disorders, e.g., reducing limbs, Severity of edema and swelling of the face, throat, upper respiratory tract, abdomen, trunk and genitals, prodromal symptoms; laryngeal swelling; non-pruritic rash; nausea; vomiting or abdominal pain.

The iRNA of the present invention can be administered as "free iRNA". Free iRNA is administered without a pharmaceutical composition. Naked iRNA can be prepared in a suitable buffer solution. The buffer solution may contain acetate, citrate, gliadin, carbonate or phosphate or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the iRNA-containing buffer solution can be adjusted to make it suitable for administration to the individual.

Alternatively, the iRNA of the present invention can be administered in a pharmaceutical composition, such as a dsRNA liposome formulation.

Individuals who benefit from suppression of AGT gene expression Individuals who are predisposed to or diagnosed with AGT-related disorders, such as hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated hypertension Hypertensive or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, and low plasma renin activity or plasma Renin concentration-related hypertension, intraocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, Unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aorta Coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth retardation, obesity, liver fat Degenerative/fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome. In one embodiment, the method includes administering a composition featured herein, thereby reducing expression of the target ab AGT gene, such as about 1, 2, 3, 4, 5, 6, 1 to 6, 1 to 3 per dose Or 3 to 6 months. In some embodiments, the composition is administered every 3 to 6 months.

In one embodiment, iRNAs useful in the methods and compositions featured herein specifically target RNAs (primary or processed) that target the AGT gene. Compositions and methods for inhibiting the expression of these genes using iRNA can be prepared and performed as described herein.

Administration of iRNA according to the methods of the invention can result in the prevention or treatment of AGT-related disorders, such as hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension Pregnancy-related hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, and low plasma renin activity or plasma renin concentration Related hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease , diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure exhaustion, myocardial infarction, angina, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic steatohepatitis ( NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome. A therapeutic amount of iRNA can be administered to an individual, such as about 0.01 mg/kg to about 200 mg/kg.

In one embodiment, the iRNA is administered subcutaneously, that is, by subcutaneous injection. One or multiple injections can be used to deliver the desired dose of iRNA to the individual. This injection can be repeated over a period of time.

Dosing can be repeated at regular intervals. In some embodiments, after an initial treatment regimen, the treatment can be administered less frequently. Repeat dosing regimens may include administration of therapeutic amounts of iRNA at regular intervals, such as once monthly to annually. In some embodiments, the iRNA is administered from about once a month to about every three months, or from about once every three months to about every six months.

The present invention also provides methods or uses of iRNA agents or pharmaceutical compositions thereof for treating individuals who would benefit from reduction and/or inhibition of AGT gene expression, for example, individuals suffering from AGT-related disorders, in combination with other drugs and/or other treatments. Methods are used in combination, for example, using known drugs and/or known treatments, such as those currently used to treat these disorders.

Accordingly, in some aspects of the invention, methods comprising administering an iRNA agent of the invention include administering one or more therapeutic agents to an individual. For example, in some embodiments, an AGT-targeting iRNA is administered in combination with: , for example, diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, alpha2-agonists agents, renin inhibitors, α-blockers, peripherally acting adrenergic drugs, selective D1 receptor partial agonists, non-selective α-adrenergic antagonists, synthetic and Steroid mineralocorticoid agent; or any combination of the foregoing, and a hypertensive treatment agent in a formulated combination. In some embodiments, the additional therapeutic agent includes an angiotensin II receptor antagonist, such as losartan, valsartan, olmesartan, eprosartan ( eprosartan) and azilsartan (azilsartan) group. In some embodiments, the additional therapeutic agent is an angiotensin receptor-neprilysin inhibitor (ARNi), e.g., Entresto®, sacubitril/valsartan; or an endothelin receptor body antagonists (ERA), for example, sitaxentan, ambrisentan, atrasentan, BQ-123, zibotentan, bosentan, horse Macitentan (macitentan), and tezosentan (tezosentan); a combination of any of the above; and a combination of a hypertension therapeutic agent formulated into a dosage form.

The iRNA and additional therapeutic agents may be administered simultaneously and/or in the same combination, e.g., parenteral or additional therapeutic agents may be administered as part of separate compositions or at different times and/or by means known in the art or as described herein. Another method of administration.

The iRNA agent and additional therapeutic agents and/or treatments may be administered simultaneously and/or in the same combination, for example, parenteral or additional therapeutic agents may be administered as part of separate compositions or at different times and/or by methods known in the art. known or administered by another method described herein.

A. Diagnostic criteria, risk factors and treatment of hypertension

Practice guidelines for the prevention and treatment of hypertension have recently been revised. Reboussin et al. (Systematic Review for the 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guidline for the Prevention,Detection,Evaluation,and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice guidlines.J Am Coll Cardiol.2017 Nov 7.pii:S0735-1097(17)41517-8.doi:10.1016/j.jacc.2017.11.004 .) and Whelton et al. (2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice guidlines.J Am Coll Cardiol.2017 Nov 7.pii:S0735-1097(17)41519-1.doi:10.1016/j.jacc.2017.11.006. ) published a large-scale report. Highlights of the new guidance are provided below. However, this guidance should be understood to provide a person of ordinary skill in the art with knowledge regarding diagnostic and monitoring standards and treatment methods for hypertension at the time of filing this application, and is incorporated herein by reference.

1.Diagnostic criteria

Although there is a persistent association between high blood pressure and increased risk of cardiovascular disease, classifying blood pressure magnitude is useful for clinical and public health decision-making. Based on average blood pressure measured in a healthcare setting (office pressure), blood pressure can be classified into 4 magnitudes: normal, elevated, and stage 1 or 2 hypertension, as shown in the table below (adapted from Whelton et al. , 2017 ).

2次情況下獲得的

2次仔細讀數的平均值的血壓。Whelton et al.,2017詳細介紹了獲得仔細血壓讀數的最佳操作，並且在所屬技術領域中係已知的。 Blood pressure is expressed in

Obtained in 2 cases

Take the average of 2 careful blood pressure readings. Best practices for obtaining careful blood pressure readings are detailed in Whelton et al., 2017, and are known in the art.

This classification is different from the classification recommended in the previous JNC7 report (Chobanian et al; the National High Blood Pressure Education Program Coordinating Committee. Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and treatment of High Blood Pressure. Hypertension. 2003 ; 42:1206-52) where stage 1 hypertension is now defined as a systolic blood pressure (SBP) of 130 to 139 or a diastolic blood pressure (DBP) of 80 to 89 mm Hg, and stage 2 hypertension in this document corresponds to stage 1 and Phase 2 JNC7 report, and the Phase 2 hypertension in this document corresponds to Phases 1 and 2 in the JNC7 report. The rationale for this classification is based on observational data on the association between SBP/DRP and cardiovascular disease risk and cardiovascular disease risk, randomized clinical trials of lifestyle modification to lower blood pressure, and treatment with antihypertensive drugs for prevention. Randomized clinical trials in cardiovascular disease.

The increased risk of cardiovascular disease in adults with stage 2 hypertension is well established. A growing number of individual studies and meta-analyses of observational data report a gradient of increasing cardiovascular disease risk from normotensive to hypertensive and stage 1 hypertension. In many of these meta-analyses, the hazard ratios for coronary heart disease and stroke were between 1.1 and 1.5, and between 1.5 and 2.0 for SBP/DBP of 120 to 129/80 to 84 mm Hg versus <120/80 mm Hg. , to compare SBP/DBP of 130 to 139/85 to 89mm Hg versus <120/80mm Hg. This risk gradient was consistent across subcategories defined by sex and race/ethnicity. The relative increase in cardiovascular disease risk associated with high blood pressure is attenuated but persists in older adults. Lifestyle modification and pharmacological antihypertensive treatment are recommended for individuals with elevated blood pressure and stage 1 and 2 hypertension. Even if blood pressure does not return to normal with treatment, clinical benefit may be obtained by reducing the period of elevated blood pressure.

2. Risk factors

Hypertension is a complex disease resulting from a combination of factors including, but not limited to, genetics, lifestyle, diet, and secondary risk factors. High blood pressure may also be related to pregnancy. It is understood that due to the complexity of hypertension, treatment of hypertension may require a variety of interventional measures. In addition, nonpharmacological interventions, including dietary and lifestyle changes, can be used to prevent and treat hypertension. Even more, intervention may provide clinical benefit without completely normalizing an individual's blood pressure.

a. Genetic risk factors

Several monogenic forms of hypertension have been identified, such as glucocorticoid-induced hyperaldosteronism, Liddle’s syndrome, Gordon’s syndrome, and others, in which single-gene mutations fully explain the pathophysiology of hypertension, and these disorders are rare. Current causes blood pressure and high The list of known genetic variations in blood pressure includes more than 25 rare mutations and 120 single nucleotide polymorphisms. However, although genetic factors may contribute to the development of hypertension in some individuals, it is estimated that genetic variation accounts for only about 3.5% of the variation in blood pressure.

b. Diet and alcohol intake

Common environmental and lifestyle risk factors for hypertension include poor diet, lack of physical activity, and excessive alcohol consumption. These factors can cause a person to become overweight or obese, further increasing the likelihood of developing or worsening high blood pressure. Increased blood pressure was more strongly associated with increased waist-to-hip ratio or other measures of central fat distribution. Obesity and persistent obesity in youth are strongly associated with hypertension in later life. Achieving a normal weight reduces the risk of high blood pressure to that of people who have never been obese.

65歲)，以及血壓量級較高或患有慢性腎病、糖尿病或代謝症候群等合併症的族群。總體而言，這些族群佔所有美國成年人的一半以上。除血壓外，鹽敏感性可能係心血管疾病和總死亡率增加的指標。目前，用於識別鹽敏感性的技術在臨床環境中係不切實際的。因此，鹽敏感性最好被視為一種族群特徵。 Intake of sodium, potassium, magnesium and calcium can also have a significant impact on blood pressure. Sodium intake is positively correlated with blood pressure and is a major contributor to age-related increases in blood pressure. Some groups are more sensitive to increased sodium intake than others, including black people and older adults (

65 years old), and those with higher blood pressure or comorbidities such as chronic kidney disease, diabetes, or metabolic syndrome. Collectively, these groups make up more than half of all U.S. adults. In addition to blood pressure, salt sensitivity may be an indicator of increased cardiovascular disease and overall mortality. Currently, techniques for identifying salt sensitivity are impractical in clinical settings. Therefore, salt sensitivity is best viewed as an ethnic trait.

Potassium intake is inversely related to blood pressure and stroke, and higher levels of potassium intake appear to balance the effects of sodium on blood pressure. Lower sodium-to-potassium ratios are associated with lower blood pressure than the corresponding sodium or potassium magnitudes themselves. Similar observations were made regarding cardiovascular disease risk.

Drinking alcohol has long been associated with high blood pressure. In the United States, alcohol consumption is estimated to account for approximately 10% of the population burden of hypertension over the long term, with the burden being greater in men than in women. Accounting for approximately 10% of the population burden of hypertension, the burden is greater in men than in women.

It should be understood that changes in diet or alcohol consumption may be part of the prevention or treatment of hypertension.

c.Physical activity

There is a well-established inverse relationship between physical activity/physical fitness and blood pressure magnitude. Even moderate levels of physical activity have been shown to be beneficial in reducing high blood pressure.

Increased physical activity is a recognized aspect of hypertension prevention or treatment.

d. Secondary risk factors

Secondary hypertension may result in severe elevations in blood pressure, pharmacologic resistance hypertension, sudden onset hypertension, elevations in blood pressure in previously controlled hypertensive patients, onset of diastolic hypertension in the elderly, and Target organ damage disproportionate to the duration or severity of hypertension. Although secondary hypertension should be suspected in young patients (<30 years) with elevated blood pressure, it is not uncommon to present with primary hypertension at younger ages, especially in blacks, as well as in some forms of secondary hypertension. Blood pressure, such as renovascular disease, is more common in older age (>65 years). Many causes of secondary hypertension are closely related to clinical manifestations or clusters of manifestations showing specific disorders. In this case, treatment of the underlying condition may resolve the raised blood pressure without administering agents typically used to treat high blood pressure.

e.Pregnancy

Pregnancy is a risk factor for hypertension, and hypertension during pregnancy is a risk factor for cardiovascular diseases and hypertension later in life. The American College of Obstetricians and Gynecologists (ACOG) published a report on pregnancy-related hypertension in 2013 (American College of Obstetricians and Gynecologists, Task Force on Hypertension in Pregnancy.Hypertension in pregnancy. Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy. Obstet Gynecol. 2013;122:1122-31). Some highlights of the report are as follows. However, this report should be understood as providing the knowledge of a person of ordinary skill in the art regarding the diagnostic and monitoring criteria and treatment of hypertension in pregnancy at the time of filing this application, and is incorporated herein by reference.

The table below provides diagnostic criteria for preeclampsia (based on Table 1 from the 2013 ACOG report).

Blood pressure management during pregnancy is complex because many commonly used antihypertensive drugs, including ACE inhibitors and ARBs, are contraindicated during pregnancy because of their potential harm to the fetus. The goals of antihypertensive treatment during pregnancy include the prevention of severe hypertension and the possibility of prolonging gestation so that the fetus has more time to mature before birth. A review of the literature on treatments for severe hypertension associated with pregnancy found insufficient evidence to recommend a specific agent; instead, clinician experience is recommended in this setting (Duley L, Meher S, Jones L. Drugs for treatment of very high blood pressure during pregnancy. Cochrane Database Syst Rev. 2013;7: CD001449.).

3. Treatment methods

Treatment of hypertension is complex because it is often associated with other comorbidities, often including reduced renal function, and individuals with reduced renal function may also be receiving treatment. Clinicians managing hypertension in adults should focus on the patient's overall health, with special emphasis on reducing the risk of adverse cardiovascular disease outcomes. All patient risk factors need to be managed in a comprehensive manner through an integrated set of non-pharmacological and pharmacological strategies. As patients' blood pressure and risk of cardiovascular disease events increase, blood pressure management should be strengthened.

2種以自不同藥理學類別的藥物以達到他們的血壓目標。了解每種劑的藥理作用機制很重要。具有互補活性的藥物方案，其中第二種抗高血壓劑係用於阻斷對初始劑的代償反應或影響不同的升壓機制，可導致附加的血壓降低。例如，噻 嗪類利尿劑可能刺激腎素-血管收縮素-醛固酮系統。藉由加入ACE抑制劑或ARB到噻嗪類，可能獲得附加的血壓降低。聯合療法的使用也可以提高依從性。有幾種2和3固定劑量藥物組合的抗高血壓藥物治療的可用，各成分之間具有互補的作用機制。 Although treatment of hypertension based solely on blood pressure magnitude and use of blood pressure-lowering medications is considered cost-effective, using a combination of absolute cardiovascular disease risk and blood pressure magnitude to guide such treatment is less effective in reducing cardiovascular risk than blood pressure alone. disease risk, more effectively and cost-effectively. Many patients who are started on a single agent will subsequently need

2 drugs from different pharmacological classes to achieve their blood pressure goals. It is important to understand the pharmacological mechanism of action of each agent. Drug regimens with complementary activity, in which a second antihypertensive agent is used to block the compensatory response to the initial agent or to affect a different pressor mechanism, may result in additive lowering of blood pressure. For example, thiazide diuretics may stimulate the renin-angiotocin-aldosterone system. By adding an ACE inhibitor or ARB to a thiazide, additional blood pressure lowering may be obtained. The use of combination therapy may also improve compliance. There are several 2- and 3-drug combinations of antihypertensive medications available, with complementary mechanisms of action between the components.

Table 18, adapted from Whelton et al. 2017, lists oral antihypertensive drugs as follows. Classes of therapeutic agents and drugs belonging to these classes are provided for the treatment of hypertension. Dosage ranges, frequencies, and comments are also provided.

Set with VIII.

In some aspects, the present disclosure provides sets of suitable containers for pharmaceutical formulations of siRNA compounds, e.g., double-stranded siRNA compounds or siRNA compounds (e.g., precursors, e.g., that can be processed into siRNA compounds or DNA encoding siRNA compounds). larger siRNA compounds, e.g., double-stranded siRNA compounds or ssiRNA compounds, or precursors thereof).

The kit includes one or more dsRNA agents and instructions for use, for example, instructions for administering a prophylactically or therapeutically effective amount of the dsRNA agent. The dsRNA agent may come in vials or prefilled syringes. The kit may optionally include a method of administering the dsRNA agent (e.g., an injection device, such as a prefilled syringe) or a method of measuring inhibition of AGT (e.g., a method of measuring inhibition of AGT mRNA, AGT protein, and/or AGT) Such methods for measuring AGT inhibition may include methods of obtaining a sample from an individual, for example, a plasma sample. The kit of the invention may optionally further include a method for determining a therapeutically or prophylactically effective amount.

In some embodiments, the individual components of the pharmaceutical formulation may be provided in a container, such as a vial or a prefilled syringe. Alternatively, if desired, the components of the pharmaceutical formulation may be provided separately in two or more containers, for example, one container for the siRNA compound formulation and at least one other container for the carrier compound. The set can be packaged in a number of different configurations, such as one or more containers in a single box. Different ingredients can be combined, for example, according to the guidelines provided in the kit. The plurality of ingredients may be combined as described herein, for example, to prepare and administer a pharmaceutical composition. The kit may also include a delivery device.

The present invention is further illustrated by the following examples, which should not be construed as being limited by the examples. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the informal sequence listings and drawings, are hereby incorporated by reference.

Example

Example 1. iRNA synthesis

Reagent source

Where the source of a reagent is not specifically given herein, such reagents may be obtained from any supplier of molecular biology reagents with quality/purity standards for molecular biology applications.

siRNA design

siRNAs targeting the human angiotensinogen (AGT) gene (human: GenBank NM_001384479.1 or NM_000029.3, NCBI gene ID: 183) were designed using customized R and Python scripts. The human NM_001384479.1 REFSEQ mRNA is 2116 bases in length and the human NM_000029.3 REFSEQ mRNA is 2587 bases in length.

A detailed list of the sense and antisense nucleotide sequences of unmodified AGT is shown in Tables 2 and 4. A detailed list of the sense and antisense nucleotide sequences of modified AGT is shown in Table 3 and Table 5.

It should be understood that throughout this application, duplex names without a decimal point are equivalent to duplex names with a decimal point, which refer only to the batch number of the duplex. For example, AD-959917 is equivalent to AD-959917.1.

siRNA synthesis

siRNA is designed, synthesized and prepared using methods known in the art.

Briefly, siRNA sequences were synthesized at 1 μmol scale using a Mermade 192 synthesizer (BioAutomation) with phosphoramidite chemistry on a solid support. The solid support is a controlled pore glass (500-1000Å) loaded with a custom GalNAc ligand (3'-GalNAc conjugate), a universal solid support (AM Chemicals), or the first nucleotide of interest. Auxiliary synthesis reagents and standard 2-cyanoethylphosphoramidite monomers (2'-deoxy-2'-fluoro, 2'-O-methyl, RNA, DNA) were obtained from Thermo-Fisher (Milwaukee, WI) , Hongene (China) or Chemgenes (Wilmington, MA, USA). Additional phosphoramidite monomer is purchased from suppliers and prepared in-house or used in custom synthesis procurement from various CMOs. Prepare phosphoramidite at a concentration of 100mM in acetonitrile or 9:1 acetonitrile:DMF and couple using 5-ethylthio-1H-tetrazole (ETT, 0.25M in acetonitrile) with a reaction time of 400 seconds . 3-((Dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, available from Chemgenes (Wilmington, Wilmington, MA, USA)) was used in Phosphorothioate linkages are generated in anhydrous acetonitrile/pyridine (9:1 v/v). The oxidation time is 5 minutes. All sequences were synthesized with the final removal of the DMT group ("DMT-Off").

After solid-phase synthesis is complete, treat solid-supported oligoribonucleotides with 300 μL of methylamine (40% aqueous solution) in a 96-well plate for approximately 2 hours at room temperature to cleave and subsequently remove from the solid-phase support All additional bases are unstable protecting groups. For sequences containing any natural ribonucleotide linkage (2'-OH) protected with a tert-butyldimethylsilyl (TBDMS) group, use TEA.3HF (triethylamine trihydrofluoride) Proceed to the second deprotection step. 200 μL of dimethylsulfoxide (DMSO) and 300 μL of TEA.3HF were added to each oligonucleotide solution in aqueous methylamine solution, and the solution was incubated at 60°C for approximately 30 minutes. After incubation, allow the plate to reach room temperature and add 1 mL of 9:1 acetonitrile:ethanol or 1:1 ethanol:isopropyl alcohol to precipitate crude oligonucleotides. The plate was then centrifuged at 4°C for 45 minutes and the supernatant was carefully decanted with the help of a multichannel pipette. The oligonucleotide pellet was redissolved in 20 mM NaOAc and subsequently used on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter, and fraction collector using a HiTrap size exclusion column (5 mL, GE Healthcare ) desalination. Desalted samples were collected in 96-well plates and analyzed by LC-MS and UV spectroscopy to confirm the identity of the material and quantify the amount of material, respectively.

Duplexing of single strands is performed on a Tecan liquid handling robot. Combine sense single-strand and antisense single-strand in 1x PBS in a 96-well plate at an equimolar ratio to a final concentration of 10 μM, seal the plate, incubate at 100°C for 10 minutes, and then incubate for 2 to 3 hours. Slowly return to room temperature within a period of time. The concentration and identity of each duplex is confirmed and then used in in vitro screening assays.

Example 2. In vitro screening method

Cell culture and 384-well transfection

Hep3b cells (ATCC, Manassas, VA) were cultured in Eagle's Minimum Essential Medium supplemented with 10% FBS (ATCC) at 37°C in an atmosphere of 5% _CO2 before being digested by trypsin and released from the well plate. (Gibco), growing close to confluence. Transfections were performed in a 384-well plate by adding 7.5 μl of Opti-MEM per well plus 0.1 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA. cat # 13778-150) to 2.5 μl of each siRNA duplex. The mixture was then incubated at room temperature for 15 minutes. Then add 40 μl of complete growth medium without antibiotics containing approximately 1.5 x10 ^cells to the siRNA mixture. Cells were incubated for 24 hours before RNA purification. Single dose experiments were performed at 10 nM, 1 nM and 0.1 nM final duplex concentrations.

Total RNA isolation using DYNABEADS mRNA isolation kit (Invitrogen ^TM , part #: 610-12)

Cells were lysed in 75 μl of lysis/binding buffer containing 3 μL of beads per well and mixed on an electrostatic shaker for 10 min. Washing steps were automated on a Biotek EL406 using magnetic plate support. Beads were washed once in buffer A (in 90 μL), once in buffer B, and twice in buffer E, with aspiration steps in between. After the last aspiration, add a complete 10 µL of RT mix to each well as described below.

Real-time PCR

In a 384-well plate (Roche cat # 04887301001), add two microliters (μl) of cDNA to a solution containing 0.5 μl human GAPDH TaqMan probe (4326317E), 0.5 μl human AGT, 2 μl nuclease-free water, and 5 μl Lightcycler 480 probe Master mix (Roche Cat # 04887301001) in each well. Real-time PCR was performed in a LightCycler480 real-time PCR system (Roche).

To calculate relative fold changes, data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. IC ₅₀ s was calculated using XLFit's 4 parameter fit model and normalized to cells transfected with AD-1955 or mock-transfected. The sense and antisense sequences of AD-1955 are as follows: sense: cuuAcGcuGAGuAcuucGAdTsdT and antisense: UCGAAGuACUcAGCGuAAGdTsdT.

Example 3. Additional duplexes targeting angiotensinogen (AGT)

Additional agents targeting the proatherosin gene were designed using custom R and Python scripts, as described below.

A detailed list of the unmodified sense and antisense nucleotide sequences of AGT is shown in Table 6. A detailed list of the modified sense and antisense nucleotide sequences of AGT is shown in Table 7.

For transfection, add 7.5 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA. cat # 13778-150) per well to 2.5 μl of each siRNA duplex in a single well of a 384-well plate. μl. The mixture was then incubated at room temperature for 15 minutes. Then add 40 μl of complete growth medium without antibiotics containing approximately 1.5 x10 ^cells to the siRNA mixture. Cells were incubated for 24 hours before RNA purification. Single dose experiments were performed at 10 nM, 1 nM and 0.1 nM final duplex concentrations.

Total RNA isolation using DYNABEADS. Briefly, cells were lysed in 75 μl of lysis/binding buffer containing 3 μL of beads per well and mixed on an electrostatic shaker for 10 min. Washing steps were automated on a Biotek EL406 using magnetic plate support. Beads were washed once in buffer A (in 3 μL), once in buffer B, and twice in buffer E, with aspiration steps in between. After the last aspiration, add a complete 12 µL of RT mix to each well as described below.

For cRNA synthesis, a master mix containing 1.5 μl of 10X buffer, 0.6 μl of 10X dNTPs, 1.5 μl of random primers, 0.75 μl of reverse transcriptase, 0.75 μl of RNase inhibitor, and 9.9 μl of H ₂ O for each reaction Add to well. The well plate was sealed, stirred on an electrostatic shaker for 10 minutes, and then incubated at 37°C for 2 hours. After this, the well plate was stirred at 80°C for 8 minutes.

RT-qPCR was performed as described above and relative fold changes were calculated. The single-dose screening results of the agents in Tables 6 and 7 in Hep3b cells are shown in Table 8.

equals

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific implementations and methods described herein. These equivalents are intended to be included within the scope of the claims below.

informal sequence listing

SEQ ID NO: 1

>gi|188595658|ref|NM_000029.3|Homo sapiens angiotensin progenitor (serine protease inhibitor, clade A, member 8) (AGT),mRNA

SEQ ID NO: 2

Reverse complement of SEQ ID NO:1

SEQ ID NO: 3

>NM_001384479.1 Homo sapiens angiotensinogen (AGT), transcript variant 1, mRNA

SEQ ID NO: 4

Reverse complement of SEQ ID NO:3

SEQ ID NO: 5

>gi|90075391|dbj|AB170313.1| Cynomolgus monkey brain cDNA selection: QmoA-10278, similar to human angiotensinogen (serine (or photocystine) protease inhibitor, clade A (α -1 antiprotease, antitrypsin), member 8) (AGT), mRNA, RefSeq: NM_000029.1

SEQ ID NO: 6

Reverse complement of SEQ ID NO:5

SEQ ID NO: 7

>gi|113461997|ref|NM_007428.3|Mouse angiotensinogen (serine protease inhibitor, peptidase inhibitor, clade A, member 8) (Agt),mRNA

SEQ ID NO: 8

Reverse complement of SEQ ID NO:7

SEQ ID NO: 9

>gi|51036672|ref|NM_134432.2|Gutter rat angiotensinogen (serine protease inhibitor, peptidase inhibitor, clade A, member 8) (Agt),mRNA

SEQ ID NO: 10

SEQ ID NO:9 reverse complement

SEQ ID NO: 11

>XM_015126038.2 PREDICTED: Macaque angiotensinogen (AGT), transcript variant X2, mRNA

SEQ ID NO: 12

Reverse complement of SEQ DI NO:11

TW202337474A_111129171_SEQL.xml

Claims (81)

A double-stranded ribonucleic acid (dsRNA) agent used to inhibit the expression of angiotensinogen (AGT) in cells, wherein, The dsRNA agent contains a sense strand and an antisense strand forming a double-stranded region; wherein, The antisense strand contains a complementary region to the mRNA encoding AGT; and wherein, The complementary region includes at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any antisense nucleotide sequence in any one of Tables 2 to 7. The dsRNA agent of claim 1, wherein the dsRNA agent contains at least 15 consecutive nucleotides and differs from any nucleotide sequence of the sense strand in any one of Tables 2 to 7 by no more than 3 nucleotide sense strands, and antisense strands containing at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any nucleotide sequence of the antisense strand in any of Tables 2 to 7 . The dsRNA agent of claim 1, wherein the dsRNA agent contains at least 15 consecutive nucleotides and differs from any nucleotide sequence of the sense strand in any one of Tables 2 to 7 by no more than 2 nucleotide sense strands, and antisense strands containing at least 15 consecutive nucleotides that differ by no more than 2 nucleotides from any nucleotide sequence of the antisense strand in any of Tables 2 to 7 . The dsRNA agent of claim 1, wherein the dsRNA agent contains at least 15 consecutive nucleotides and differs from any nucleotide sequence of the sense strand in any one of Tables 2 to 7 by no more than 1 nucleotide sense strands, and antisense strands containing at least 15 consecutive nucleotides that differ by no more than 1 nucleotide from any nucleotide sequence of the antisense strand in any of Tables 2 to 7 . The dsRNA agent according to claim 1, wherein the dsRNA agent contains a nucleotide sequence selected from the group consisting of any nucleotide sequence of the sense strand in any one of Tables 2 to 7. The sense strand; and an antisense strand comprising a nucleotide sequence selected from the group consisting of any nucleotide sequence of the antisense strand in any one of Tables 2 to 7. The dsRNA agent of claim 1, wherein the dsRNA agent contains at least one modified nucleotide. The dsRNA agent of claim 1 or 6, wherein substantially all the nucleotides of the sense strand are modified nucleotides; substantially all the nucleotides of the antisense strand are modified nucleotides; or substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides. The dsRNA agent as described in claim 1, 6 or 7, wherein all the nucleotides of the sense strand are modified nucleotides; all the nucleotides of the antisense strand are modified nucleosides. acid; or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides. The dsRNA agent according to any one of claims 6 to 8, wherein at least one modified nucleotide is selected from the group consisting of: deoxynucleotide, 3'-terminal deoxythymine (dT) Nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, unlocked core Glycosides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl Base-modified nucleotides, N-morpholine nucleotides, aminophosphates, non-natural bases including nucleotides, tetrahydropyran-modified nucleotides, 1,5-hydrohexitol modification of nucleosides Acid, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, 5'-phosphate containing nucleotides, 5 Nucleotides that are '-phosphate mimetics, vinylphosphonate nucleotides, thermally destabilized nucleotides, glycol-modified nucleotides (GNA), nucleotides containing 2' phosphate, and 2- O-(N-methylacetamide) modified nucleotides; and combinations thereof. The dsRNA agent according to any one of claims 6 to 8, wherein the at least one modified nucleotide is selected from the group consisting of: LNA, HNA, CeNA, 2' -methoxyethyl base, 2' - O-alkyl, 2' - O-allyl, 2' - C-allyl, 2' -fluoro, 2' -deoxy, 2'-hydroxy and ethylene glycol; and combination. The dsRNA agent according to any one of claims 6 to 8, wherein the at least one modified nucleotide is selected from the group consisting of: deoxy-nucleotide, 2'-O-methyl base-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, glycol-modified nucleotides (GNA), nucleotides containing 2' phosphates and nucleotides containing a phosphorothioate group; and combinations thereof. The dsRNA agent according to any one of claims 6 to 8, wherein the at least one modified nucleotide is a nucleotide modified with a thermally unstable nucleotide. The dsRNA agent of claim 12, wherein the heat-labile nucleotide modification is selected from the group consisting of: no base modification; mismatch with the opposite nucleotide in the duplex; Stable sugar modification, 2'-deoxy modification, acyclic nucleotide, unlocked nucleic acid (UNA) and glyceryl nucleic acid (GNA). The dsRNA agent according to any one of claims 1 to 13, wherein the double-stranded region is 19 to 30 nucleotide pairs in length. The dsRNA agent of claim 14, wherein the double-stranded region is 19 to 25 nucleotide pairs in length. The dsRNA agent of claim 14, wherein the double-stranded region is 19 to 23 nucleotide pairs in length. The dsRNA agent of claim 14, wherein the double-stranded region is 23 to 27 nucleotide pairs in length. The dsRNA agent of claim 14, wherein the double-stranded region is 21 to 23 nucleotide pairs in length. The dsRNA agent of any one of claims 1 to 18, wherein the strands are each independently no more than 30 nucleotides in length. The dsRNA agent of any one of claims 1 to 19, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. The dsRNA agent of any one of claims 1 to 20, wherein the complementary region is at least 17 nucleotides in length. The dsRNA agent according to any one of claims 1 to 21, wherein the complementary region is at least 19 to 23 nucleotides in length. The dsRNA agent according to any one of claims 1 to 22, wherein the complementary region is 19 nucleotides in length. The dsRNA agent of any one of claims 1 to 23, wherein the at least one strand comprises a 3' overhang of at least 1 nucleotide. The dsRNA agent of any one of claims 1 to 23, wherein the at least one strand comprises a 3' overhang of at least 2 nucleotides. The dsRNA agent according to any one of claims 1 to 25, further comprising a ligand. The dsRNA agent according to claim 26, wherein the ligand is connected to the 3' end of the sense strand of the dsRNA agent. The dsRNA agent according to claim 26 or 27, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative. The dsRNA agent according to any one of claims 26 to 28, wherein the ligand is one or more GalNAc derivatives attached by a monovalent, divalent or trivalent branched linker. The dsRNA agent according to claim 28 or 29, wherein the ligand is

The dsRNA agent of claim 30, wherein the dsRNA agent is coupled to the ligand as follows:

And, among them, X is O or S. The dsRNA agent of claim 31, wherein the X is O. The dsRNA agent of any one of claims 1 to 32, wherein the dsRNA agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. The dsRNA agent of claim 33, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3'-terminus of one strand. The dsRNA agent according to claim 34, wherein the strand is an antisense strand. The dsRNA agent according to claim 34, wherein the strand is a sense strand. The dsRNA agent of claim 33, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5'-terminus of one strand. The dsRNA agent according to claim 37, wherein the strand is an antisense strand. The dsRNA agent as described in claim 37, wherein the stock is a sense stock. The dsRNA agent of claim 33, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5'- and 3'-termini of one strand. The dsRNA agent according to claim 40, wherein the strand is an antisense strand. The dsRNA agent according to any one of claims 1 to 41, wherein the base pair located at the 5' -terminal 1 position of the antisense strand of the duplex is an AU base pair. A cell comprising the dsRNA agent according to any one of claims 1 to 42. A pharmaceutical composition for inhibiting the expression of a gene encoding angiotensinogen (AGT), including the dsRNA agent described in any one of claims 1 to 42 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 44, wherein the dsRNA agent is in a buffer-free solution. The pharmaceutical composition according to claim 45, wherein the buffer-free solution is saline or water The pharmaceutical composition according to claim 44, wherein the dsRNA agent is in a buffer solution. The pharmaceutical composition of claim 47, wherein the buffer solution contains acetate, citrate, prolamin, carbonate or phosphate or any combination thereof. The pharmaceutical composition according to claim 48, wherein the buffer solution is phosphate buffered saline (PBS). A method for inhibiting angiotensinogen (AGT) gene expression in cells, the method comprising contacting cells with the dsRNA agent described in any one of claims 1 to 42 or the dsRNA agent described in any one of claims 44 to 49 Contact with pharmaceutical compositions thereby inhibits the expression of the AGT gene in cells. The method of claim 50, wherein the cell line is within an individual. The method of claim 51, wherein the system is human. The method of claim 51, wherein the individual has an AGT-related disorder. The method of claim 53, wherein the AGT-related disorder is selected from the group consisting of: hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated hypertension Systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, and low plasma renin activity or plasma renin concentration-related hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, Arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, Stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease Liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome. The method of claim 54, wherein the subject has a systolic blood pressure of at least 130 mm Hg or a diastolic blood pressure of at least 80 mm Hg. The method of claim 54, wherein the subject has a systolic blood pressure of at least 130 mm Hg and a diastolic blood pressure of at least 80 mm Hg. The method of claim 54, wherein the system is part of a population susceptible to salt sensitivity, is overweight, is obese, or is pregnant. The method of any one of claims 50 to 57, wherein the cells are contacted with a dsRNA agent to inhibit the expression of AGT by at least 50%, 60%, 70%, 80%, 90% or 95%. The method of any one of claims 50 to 58, wherein AGT expression is inhibited to reduce AGT protein levels in the individual's serum by at least 50%, 60%, 70%, 80%, 90% or 95%. A method of treating an individual suffering from a disorder who would benefit from reduced expression of angiotensinogen (AGT), comprising adding a therapeutically effective amount of a dsRNA agent of any one of claims 1 to 42 or claims 44 to 49 The pharmaceutical composition of any one of the above is administered to the individual, thereby treating the individual suffering from the disorder and who would benefit from reduced expression of AGT. A method of preventing at least one symptom in an individual suffering from a disorder who would benefit from reduced expression of angiotensinogen (AGT), comprising a prophylactically effective amount of a dsRNA agent of any one of claims 1 to 42, or The pharmaceutical composition of any one of claims 44 to 49 is administered to the individual to prevent at least one symptom in the individual suffering from the disorder and who would benefit from a reduction in AGT performance. The method of claim 60 or 61, wherein the disorder is an AGT-related disorder. The method of claim 62, wherein the AGT-related disorder is selected from the group consisting of: hypertensive hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated hypertension Systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, and low plasma renin activity or Hypertension and hyperphthalmia related to plasma renin concentration pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy , chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, renal disease, renal failure, systemic sclerosis disease, intrauterine fetal growth retardation (IUGR), fetal growth retardation, obesity, hepatic steatosis/fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, 2 Type 2 diabetes (non-insulin-dependent diabetes) and metabolic syndrome. The method of claim 63, wherein the subject has a systolic blood pressure of at least 130 mm Hg or a diastolic blood pressure of at least 80 mm Hg. The method of claim 63, wherein the subject has a systolic blood pressure of at least 130 mm Hg and a diastolic blood pressure of at least 80 mm Hg. The method of claim 63, wherein the system is part of a population susceptible to salt sensitivity, is overweight, is obese, or is pregnant. A method as claimed in any one of claims 60 to 66, wherein the system is human. The method of any one of claims 60 to 67, wherein administration of the dsRNA agent to the individual results in a reduction in AGT protein accumulation in the individual. The method of any one of claims 60 to 68, wherein the dsRNA agent is administered to the individual at a dose of about 0.01 mg/kg to 50 mg/kg. The method of any one of claims 60 to 69, wherein the dsRNA agent is administered to the individual subcutaneously. The method of any one of claims 60 to 70, further comprising determining the AGT level in a sample from the individual. The method of claim 71, wherein the AGT level in the individual sample is the AGT protein level in the blood or serum or urine or liver tissue sample. The method of any one of claims 60 to 72, further comprising determining the bradylin level, the protonin level or the blood pressure of the individual. The method of any one of claims 60 to 73, comprising administering to the individual an additional therapeutic agent for a method of treating an AGT-related disorder. The method of claim 74, wherein the additional therapeutic agent is selected from the group consisting of: diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers Interceptors, vasodilators, calcium channel blockers, aldosterone antagonists, α2-agonists, renin inhibitors, α-blockers, peripherally acting adrenergic drugs, selective D1 receptor partial agonists , non-selective alpha-adrenergic antagonists, synthetics, steroid mineralocorticoids, angiotensin receptor-neprilysin inhibitor (ARNi), Entresto®, sacubitril/valsartan; or endothelin receptor antagonist (ERA), sitaxentan, ambrisentan, atrasentan, BQ-123, zibotentan, bosentan , macitentan (macitentan) and tezosentan (tezosentan); a combination of any of the above; and a hypertension therapeutic agent formulated as a combination of agents. The method of claim 74, wherein the additional therapeutic agent comprises an angiotensin II receptor antagonist. The method of claim 76, wherein the angiotensin II receptor antagonist is selected from the group consisting of: losartan, valsartan, olmesartan, eprosartan and azilsartan. A kit comprising the dsRNA agent according to any one of claims 1 to 42 or the pharmaceutical composition according to any one of claims 44 to 49. A vial containing the dsRNA agent as described in any one of claims 1 to 42 or the pharmaceutical composition as described in any one of claims 44 to 49. A syringe containing the dsRNA agent according to any one of claims 1 to 42 or the pharmaceutical composition according to any one of claims 44 to 49. An RNA-induced silencing complex (RISC) comprising an antisense strand of the dsRNA agent according to any one of claims 1 to 42.